The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages.

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.

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.

The glucocorticoid-induced leucine zipper mediates statin-induced muscle damage.

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.

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

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) 1ß, 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.

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.

Glucocorticoid-induced leucine zipper: a critical factor in macrophage endotoxin tolerance.

Induction of glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids plays a key role in their anti-inflammatory action. In activated macrophages, GILZ levels are downregulated via tristetraprolin-mediated GILZ mRNA destabilization. To assess the functional significance of GILZ downregulation, we generated myeloid-specific GILZ knockout (KO) mice. GILZ-deficient macrophages displayed a higher responsiveness toward LPS, as indicated by increased TNF-α and IL-1ß expression. This effect was due to an activation of ERK, which was significantly amplified in GILZ KO cells. The LPS-induced activation of macrophages is attenuated upon pretreatment of macrophages with low-dose LPS, an effect termed endotoxin tolerance. In LPS-tolerant macrophages, GILZ mRNA was stabilized, whereas ERK activation was strongly decreased. In contrast, GILZ KO macrophages exhibited a strongly reduced desensitization. To explore the contribution of GILZ expression in macrophages to endotoxin tolerance in vivo, we treated GILZ KO mice with repeated i.p. injections of low-dose LPS followed by treatment with high-dose LPS. LPS pretreatment resulted in reduced proinflammatory mediator expression upon high-dose LPS treatment in serum and tissues. In contrast, cytokine induction was preserved in tolerized GILZ KO animals. In summary, our data suggest that GILZ is a key regulator of macrophage functions.

Metabolic Profiling of SH-SY5Y and Neuro2A Cells in Relation to Fetal Calf Serum (FCS) Concentration in Culture Media.

The neuroblastoma cell lines SH-SY5Y and Neuro2A are commonly utilized models in neurobiological research. DMEM supplemented with different nutrients and 5-10% Fetal Calf Serum (FCS) is typically used for culturing these cell lines. During special treatments, a reduced FCS content is often deployed to reduce cellular proliferation or the content of bioactive compounds. The impact of the reduction of FCS in culture media on the metabolic profile of SH-SY5Y and Neuro2A cells is currently unknown. Using an Amplex Red Assay, this study showed that the consumption of L-glutamine decreased after FCS reduction. Glucose and pyruvate consumption increased in both cell lines after the reduction of FCS. Thus, lactate production also increased with reduced FCS concentration. The reduction of FCS in the cell culture medium resulted in a reduced aerobic ATP production for SH-SY5Y cells and a complete shut down of aerobic ATP production for Neuro2A cells, measured using the Seahorse XF Real-Time ATP Rate Assay. Utilizing the Seahorse XF Glutamine Oxidation Stress Test, Neuro2A cells showed an increased utilization of L-glutamine oxidation after reduction of FCS. These results indicate that changes in FCS concentration in culture media have an impact on the different energy production strategies of SH-SY5Y and Neuro2A cells which must be considered when planning special treatments.

A cell-free, biomimetic hydrogel based on probiotic membrane vesicles ameliorates wound healing.

Probiotic bacteria, such as Lactobacilli, have been shown to elicit beneficial effects in various tissue regeneration applications. However, their formulation as living bacteria is challenging, and their therapeutic use as proliferating microorganisms is especially limited in immunocompromised patients. Here, we propose a new therapeutic avenue to circumvent these shortcomings by developing a bacteriomimetic hydrogel based on membrane vesicles (MVs) produced by Lactobacilli. We coupled MVs from Lactobacillus plantarum and Lactobacillus casei, respectively, to the surface of synthetic microparticles, and embedded those bacteriomimetics into a pharmaceutically applicable hydrogel matrix. The wound microenvironment changes during the wound healing process, including adaptions of the pH and changes of the oxygen supply. We thus performed proteomic characterization of the MVs harvested under different culture conditions and identified characteristic proteins related to the biological effect of the probiotics in every culture state. In addition, we highlight a number of unique proteins expressed and sorted into the MVs for every culture condition. Using different in vitro models, we demonstrated that increased cell migration and anti-inflammatory effects of the bacteriomimetic microparticles were dependent on the culture condition of the secreting bacteria. Finally, we demonstrated the bacteriomimetic hydrogel's ability to improve healing in an in vivo mouse full-thickness wound model. Our results create a solid basis for the future application of probiotic-derived vesicles in the treatment of inflammatory dispositions and stimulates the initiation of further preclinical trials.

Immunomodulation by glucocorticoid-induced leucine zipper in macrophages: enhanced phagocytosis, protection from pyroptosis, and altered mitochondrial function.

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.

C-Myc/H19/miR-29b axis downregulates nerve/glial (NG)2 expression in glioblastoma multiforme.

Nerve/glial antigen (NG)2 is highly expressed in glioblastoma multiforme (GBM). However, the underlying mechanisms of its upregulated expression are largely unknown. In silico analyses reveal that the tumor-suppressive miR-29b targets NG2. We used GBM-based data from The Cancer Genome Atals databases to analyze the expression pattern of miR-29b and different target genes, including NG2. Moreover, we investigated the regulatory function of miR-29b on NG2 expression and NG2-related signaling pathways. We further studied upstream mechanisms affecting miR-29b-dependent NG2 expression. We found that miR-29b downregulates NG2 expression directly and indirectly via the transcription factor Sp1. Furthermore, we identified the NG2 coreceptor platelet-derived growth factor receptor (PDGFR)α as an additional miR-29b target. As shown by a panel of functional cell assays, a reduced miR-29b-dependent NG2 expression suppresses tumor cell proliferation and migration. Signaling pathway analyses revealed that this is associated with a decreased ERK1/2 activity. In addition, we found that the long noncoding RNA H19 and c-Myc act as upstream repressors of miR-29b in GBM cells, resulting in an increased NG2 expression. These findings indicate that the c-Myc/H19/miR-29b axis crucially regulates NG2 expression in GBM and, thus, represents a target for the development of future GBM therapies.

Gene Editing and Small Molecule Inhibitors of the RNA Binding Protein IGF2BP2/IMP2 Show its Potential as an Anti-Cancer Drug Target.

BACKGROUND: The RNA-binding protein IGF2BP2/IMP2/VICKZ2/p62 is an oncofetal protein that is overexpressed in several cancer entities. Employing IMP2 knockout colorectal cancer cells, we could show the important role of IMP2 in several hallmarks of cancer. This study aimed to functionally characterize IMP2 in lung (A549, LLC1) and hepatocellular carcinoma (HepG2, Huh7) cell lines to assess its role as a potential target for these cancer entities. METHODS: IMP2 knockouts were generated by CRISPR/Cas9 and its variant approach prime editing; the editing efficiency of two single guide RNAs (sgRNAs) was verified via next-generation sequencing. We studied the effect of IMP2 knockout on cell proliferation, colony formation, and migration and employed small-molecule inhibitors of IMP2. RESULTS: Despite multiple attempts, it was not possible to generate IMP2 biallelic knockouts in A549 and Huh7 cells. Both sgRNAs showed good editing efficiency. However, edited cells lost their ability to proliferate. The attempt to generate an IMP2 biallelic knockout in LLC1 cells using CRISPR/Cas9 was successful. Monoallelic knockout cell lines of IMP2 showed a reduction in 2D cell proliferation and reduced migration. In 3D cultures, a change in morphology from compact spheroids to loose aggregates and a distinct reduction in the colony formation ability of the IMP2 knockouts was observed, an effect that was mimicked by previously identified IMP2 inhibitor compounds that also showed an inhibitory effect on colony formation. CONCLUSIONS: Our in vitro target validation supports that IMP2 is essential for tumor cell proliferation, migration, and colony formation in several cancer entities.

Glucocorticoid-induced leucine zipper is downregulated in human alveolar macrophages upon Toll-like receptor activation.

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.

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.

Alterations in the hepatocyte epigenetic landscape in steatosis.

Fatty liver disease or the accumulation of fat in the liver, has been reported to affect the global population. This comes with an increased risk for the development of fibrosis, cirrhosis, and hepatocellular carcinoma. Yet, little is known about the effects of a diet containing high fat and alcohol towards epigenetic aging, with respect to changes in transcriptional and epigenomic profiles. In this study, we took up a multi-omics approach and integrated gene expression, methylation signals, and chromatin signals to study the epigenomic effects of a high-fat and alcohol-containing diet on mouse hepatocytes. We identified four relevant gene network clusters that were associated with relevant pathways that promote steatosis. Using a machine learning approach, we predict specific transcription factors that might be responsible to modulate the functionally relevant clusters. Finally, we discover four additional CpG loci and validate aging-related differential CpG methylation. Differential CpG methylation linked to aging showed minimal overlap with altered methylation in steatosis.

Spray-dried pneumococcal membrane vesicles are promising candidates for pulmonary immunization.

Pneumococcal infections represent a global health threat, which requires novel vaccine developments. Extracellular vesicles are secreted from most cells, including prokaryotes, and harbor virulence factors and antigens. Hence, bacterial membrane vesicles (MVs) may induce a protective immune response. For the first time, we formulate spray-dried gram-positive pneumococcal MVs-loaded vaccine microparticles using lactose/leucine as inert carriers to enhance their stability and delivery for pulmonary immunization. The optimized vaccine microparticles showed a mean particle size of 1-2 µm, corrugated surface, and nanocrystalline nature. Their aerodynamic diameter of 2.34 µm, average percentage emitted dose of 88.8%, and fine powder fraction 79.7%, demonstrated optimal flow properties for deep alveolar delivery using a next-generation impactor. Furthermore, confocal microscopy confirmed the successful encapsulation of pneumococcal MVs within the prepared microparticles. Human macrophage-like THP-1 cells displayed excellent viability, negligible cytotoxicity, and a rapid uptake around 60% of fluorescently labeled MVs after incubation with vaccine microparticles. Moreover, vaccine microparticles increased the release of pro-inflammatory cytokines tumor necrosis factor and interleukin-6 from primary human peripheral blood mononuclear cells. Vaccine microparticles exhibited excellent properties as promising vaccine candidates for pulmonary immunization and are optimal for further animal testing, scale-up and clinical translation.

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.

Nano-in-Microparticles for Aerosol Delivery of Antibiotic-Loaded, Fucose-Derivatized, and Macrophage-Targeted Liposomes to Combat Mycobacterial Infections: In Vitro Deposition, Pulmonary Barrier Interactions, and Targeted Delivery.

Nontuberculous mycobacterial infections rapidly emerge and demand potent medications to cope with resistance. In this context, targeted loco-regional delivery of aerosol medicines to the lungs is an advantage. However, sufficient antibiotic delivery requires engineered aerosols for optimized deposition. Here, the effect of bedaquiline-encapsulating fucosylated versus nonfucosylated liposomes on cellular uptake and delivery is investigated. Notably, this comparison includes critical parameters for pulmonary delivery, i.e., aerosol deposition and the noncellular barriers of pulmonary surfactant (PS) and mucus. Targeting increases liposomal uptake into THP-1 cells as well as peripheral blood monocyte- and lung-tissue derived macrophages. Aerosol deposition in the presence of PS, however, masks the effect of active targeting. PS alters antibiotic release that depends on the drug's hydrophobicity, while mucus reduces the mobility of nontargeted more than fucosylated liposomes. Dry-powder microparticles of spray-dried bedaquiline-loaded liposomes display a high fine particle fraction of >70%, as well as preserved liposomal integrity and targeting function. The antibiotic effect is maintained when deposited as powder aerosol on cultured Mycobacterium abscessus. When treating M. abscessus infected THP-1 cells, the fucosylated variant enabled enhanced bacterial killing, thus opening up a clear perspective for the improved treatment of nontuberculous mycobacterial infections.

Dysregulation of cholesterol homeostasis in human lung cancer tissue and tumour-associated macrophages.

BACKGROUND: Based on reports on elevated cholesterol levels in cancer cells, strategies to lower cholesterol synthesis have been suggested as an antitumour strategy. However, cholesterol depletion has also been shown to induce tumour-promoting actions in tumour-associated macrophages (TAMs). METHODS: We performed lipidomic and transcriptomic analyses of human lung cancer material. To assess whether the TAM phenotype is shaped by secreted factors produced by tumour cells, primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype using tumour cell-conditioned medium. FINDINGS: Lipidomic analysis of lung adenocarcinoma (n=29) and adjacent non-tumour tissues (n=22) revealed a significant accumulation of free cholesterol and cholesteryl esters within the tumour tissue. In contrast, cholesterol levels were reduced in TAMs isolated from lung adenocarcinoma tissues when compared with alveolar macrophages (AMs) obtained from adjacent non-tumour tissues. Bulk-RNA-Seq revealed that genes involved in cholesterol biosynthesis and metabolism were downregulated in TAMs, while cholesterol efflux transporters were upregulated. In vitro polarized TAM-like macrophages showed an attenuated lipogenic gene expression signature and exhibited lower cholesterol levels compared with non-polarized macrophages. A genome-wide comparison by bulk RNA-Seq confirmed a high similarity of ex vivo TAMs and in vitro TAM-like macrophages. Modulation of intracellular cholesterol levels by either starving, cholesterol depletion, or efflux transporter inhibition indicated that cholesterol distinctly shapes macrophage gene expression. INTERPRETATION: Our data show an opposite dysregulation of cholesterol homeostasis in tumour tissue vs. TAMs. Polarization of in vitro differentiated macrophages by tumour cell-conditioned medium recapitulates key features of ex vivo TAMs. FUNDING: Deutsche Forschungsgemeinschaft (DFG), Landesforschungsf €orderungsprogramm Saarland (LFPP).

Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages.

BACKGROUND: Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. METHODS: Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. RESULTS: IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6. CONCLUSION: AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.