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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.
Background: Investigations on pulmonary macrophages (MF) mostly focus on alveolar MF (AM) as a well-defined cell population. Characteristics of MF in the interstitium, referred to as lung interstitial MF (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 MF 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 MF in the inflammatory response.
Abstract Nanoparticles (NPs) are able to deliver a variety of substances into eukaryotic cells. However, their usage is often hampered by a lack of specificity, leading to the undesired uptake of NPs by virtually all cell types. In contrast to this, yeast is known to be specifically taken up into immune cells after entering the body. Therefore, we investigated the interaction of biodegradable surface-modified poly (lactic-co-glycolic acid) (PLGA) particles with yeast cells to overcome the unspecificity of the particulate carriers. Cells of different Saccharomyces cerevisiae strains were characterized regarding their interaction with PLGA-NPs under isotonic and hypotonic conditions. The particles were shown to efficiently interact with yeast cells leading to stable NP/yeast-complexes allowing to associate or even internalize compounds. Notably, applying those complexes to a co-culture model of HeLa cells and macrophages, the macrophages were specifically targeted. This novel nano-in-micro carrier system suggests itself as a promising tool for the delivery of biologically active agents into phagocytic cells combining specificity and efficiency. This article is protected by copyright. All rights reserved.
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.
In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
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.
Abstract 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.
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.
A cell-free, biomimetic hydrogel based on probiotic membrane vesicles ameliorates wound healing
(2024)
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.
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 data sets 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.