Glucocorticoids are potent anti-inflammatory drugs, although their use is associated with severe side effects. Loading glucocorticoids into suitable nanocarriers can significantly reduce these undesirable effects. Macrophages play a crucial role in inflammation, making them strategic targets for glucocorticoid-loaded nanocarriers. The main objective of this study is to develop a glucocorticoid-loaded PLGA nanocarrier specifically targeting liver macrophages, thereby enabling the localized release of glucocorticoids at the site of inflammation. Dexamethasone acetate (DA)-loaded PLGA nanospheres designed for passive macrophage targeting are synthesized using the nanoprecipitation method. Two types of PLGA NSs in the size range of 100-300 nm are prepared, achieving a DA-loading efficiency of 19 %. Sustained DA release from nanospheres over 3 days is demonstrated. Flow cytometry analysis using murine bone marrow-derived macrophages demonstrates the efficient internalization of fluorescent dye-labeled PLGA nanospheres, particularly into pro-inflammatory macrophages. Significant down-regulation in pro-inflammatory cytokine genes mRNA is observed without apparent cytotoxicity after treatment with DA-loaded PLGA nanospheres. Subsequent experiments in mice confirm liver macrophage-specific nanospheres accumulation following intravenous administration using in vivo imaging, flow cytometry, and fluorescence microscopy. Taken together, the data show that the DA-loaded PLGA nanospheres are a promising drug-delivery system for the treatment of inflammatory liver diseases.
- MeSH
- antiflogistika farmakologie chemie MeSH
- dexamethason * farmakologie chemie analogy a deriváty MeSH
- játra * účinky léků metabolismus MeSH
- kopolymer kyseliny glykolové a mléčné * chemie MeSH
- makrofágy * účinky léků metabolismus MeSH
- myši MeSH
- nanokuličky * chemie MeSH
- nosiče léků chemie farmakologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related nuclear receptors with overlapping regulatory functions in xenobiotic clearance but distinct roles in endobiotic metabolism. Car activation has been demonstrated to ameliorate hypercholesterolemia by regulating cholesterol metabolism and bile acid elimination, whereas PXR activation is associated with hypercholesterolemia and liver steatosis. Here we show a human CAR agonist/PXR antagonist, MI-883, which effectively regulates genes related to xenobiotic metabolism and cholesterol/bile acid homeostasis by leveraging CAR and PXR interactions in gene regulation. Through comprehensive analyses utilizing lipidomics, bile acid metabolomics, and transcriptomics in humanized PXR-CAR-CYP3A4/3A7 mice fed high-fat and high-cholesterol diets, we demonstrate that MI-883 significantly reduces plasma cholesterol levels and enhances fecal bile acid excretion. This work paves the way for the development of ligands targeting multiple xenobiotic nuclear receptors. Such ligands hold the potential for precise modulation of liver metabolism, offering new therapeutic strategies for metabolic disorders.
- MeSH
- cholesterol * metabolismus krev MeSH
- cytochrom P-450 CYP3A metabolismus genetika MeSH
- dieta s vysokým obsahem tuků * škodlivé účinky MeSH
- hypercholesterolemie * farmakoterapie metabolismus MeSH
- hypolipidemika farmakologie terapeutické užití MeSH
- játra metabolismus účinky léků MeSH
- konstitutivní androstanový receptor * MeSH
- lidé MeSH
- metabolismus lipidů účinky léků MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- pregnanový X receptor * metabolismus genetika MeSH
- pyridiny MeSH
- receptory cytoplazmatické a nukleární * metabolismus agonisté genetika MeSH
- regulace genové exprese účinky léků MeSH
- žlučové kyseliny a soli * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Francisella tularensis secretes tubular outer membrane vesicles (OMVs) that contain a number of immunoreactive proteins as well as virulence factors. We have reported previously that isolated Francisella OMVs enter macrophages, cumulate inside, and induce a strong pro-inflammatory response. In the current article, we present that OMVs treatment of macrophages also enhances phagocytosis of the bacteria and suppresses their intracellular replication. On the other hand, the subsequent infection with Francisella is able to revert to some extent the strong pro-inflammatory effect induced by OMVs in macrophages. Being derived from the bacterial surface, isolated OMVs may be considered a "non-viable mixture of Francisella antigens" and as such, they present a promising protective material. Immunization of mice with OMVs isolated from a virulent F. tularensis subsp. holarctica strain FSC200 prolonged the survival time but did not fully protect against the infection with a lethal dose of the parent strain. However, the sera of the immunized animals revealed unambiguous cytokine and antibody responses and proved to recognize a set of well-known Francisella immunoreactive proteins. For these reasons, Francisella OMVs present an interesting material for future protective studies.
- Publikační typ
- časopisecké články MeSH
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.
The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.
- MeSH
- hepatocyty účinky léků metabolismus MeSH
- konstitutivní androstanový receptor * agonisté chemie MeSH
- lidé MeSH
- myši MeSH
- pyridiny farmakologie MeSH
- receptory cytoplazmatické a nukleární metabolismus MeSH
- steroidní receptory * agonisté chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Affinity purification, combined with mass spectrometry (AP-MS) is considered a pivotal technique in protein-protein interaction studies enabling systematic detection at near physiological conditions. The addition of a quantitative proteomic method, like SILAC metabolic labeling, allows the elimination of non-specifically bound contaminants which greatly increases the confidence of the identified interaction partners. Compared to eukaryotic cells, the SILAC labeling of bacteria has specificities that must be considered. The protocol presented here describes the labeling of bacterial cultures with stable isotope-labeled amino acids, purification of an affinity-tagged protein, and sample preparation for MS measurement. Finally, we discuss the analysis and interpretation of MS data to identify and select the specific partners interacting with the protein of interest. As an example, this workflow is applied to the discovery of potential interaction partners of glyceraldehyde-3-phosphate dehydrogenase in the gram-negative bacterium Francisella tularensis.
Francisella tularensis is a highly infectious Gram-negative coccobacillus which causes the disease tularemia. The potential for its misuse as a biological weapon has led disease control and prevention centers to classify this bacterium as a category A agent. Bacterial outer membrane vesicles (OMVs) are spherical particles 20-250 nm in size produced by all Gram-negative bacteria and constitute one of the major secretory pathways. Bacteria use them in interacting with both other bacterial cells and eukaryotic (host) cells. OMVs of Francisella contain number of its so far described virulence factors and immunomodulatory proteins. Their role in host-pathogen interactions can therefore be presumed, and the possibility exists also for their potential use in a subunit vaccine. Moreover, Francisella microbes produce both usual spherical and unusual tubular OMVs. Because OMVs emerge from the outermost surface of the bacterial cell, we focused on the secretion of OMVs in several mutant Francisella strains with disrupted surface structures (namely the O-antigen). O-antigen in Francisella is not only the structural component of LPS but also forms another important virulence factor: the O-antigen polysaccharide capsule. Mutant strain phenotypes were evaluated by growth curves, vesiculation rates, their sensitivity to the complement contained in serum, and proliferation inside murine bone marrow macrophages. Morphologies of both OMVs and the bacteria were visualized by electron microscopy. The O-antigen mutant strains were considerably attenuated in serum resistance and intracellular proliferation. All the strains showed lower ability to form the tubular OMVs. Some strains formed tubular protrusions from their outer membrane but their stability was weak. Some hypervesiculating strains were revealed that will serve as source of OMVs for further studies of their protective potential. Our results suggest the presence of LPS and the O-antigen capsule on the surface of Francisella to be critical not only for its virulence but also for the exceptional tubular shape of its OMVs.
- MeSH
- Francisella tularensis * genetika MeSH
- gramnegativní bakterie MeSH
- lipopolysacharidy chemie MeSH
- myši MeSH
- O-antigeny MeSH
- proteiny vnější bakteriální membrány genetika metabolismus MeSH
- tularemie * mikrobiologie prevence a kontrola MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Francisella tularensis is known to release unusually shaped tubular outer membrane vesicles (OMV) containing a number of previously identified virulence factors and immunomodulatory proteins. In this study, we present that OMV isolated from the F. tularensis subsp. holarctica strain FSC200 enter readily into primary bone marrow-derived macrophages (BMDM) and seem to reside in structures resembling late endosomes in the later intervals. The isolated OMV enter BMDM generally via macropinocytosis and clathrin-dependent endocytosis, with a minor role played by lipid raft-dependent endocytosis. OMVs proved to be non-toxic and had no negative impact on the viability of BMDM. Unlike the parent bacterium itself, isolated OMV induced massive and dose-dependent proinflammatory responses in BMDM. Using transmission electron microscopy, we also evaluated OMV release from the bacterial surface during several stages of the interaction of Francisella with BMDM. During adherence and the early phase of the uptake of bacteria, we observed numerous tubular OMV-like protrusions bulging from the bacteria in close proximity to the macrophage plasma membrane. This suggests a possible role of OMV in the entry of bacteria into host cells. On the contrary, the OMV release from the bacterial surface during its cytosolic phase was negligible. We propose that OMV play some role in the extracellular phase of the interaction of Francisella with the host and that they are involved in the entry mechanism of the bacteria into macrophages.
- Publikační typ
- časopisecké články MeSH
Release of outer membrane vesicles (OMV) is an important phenomenon in Gram-negative bacteria playing multiple roles in their lifestyle, including in relation to virulence and host-pathogen interaction. Francisella tularensis, unlike other bacteria, releases unusually shaped, tubular OMV. We present a proteomic comparison of OMV and membrane fractions from two F. tularensis strains: moderately virulent subsp. holarctica strain FSC200 and highly virulent subsp. tularensis strain SchuS4. Proteomic comparison studies routinely evaluate samples from the same proteome, but sometimes we must compare samples from closely related organisms. This raises quantification issues. We propose a novel approach to cross-species proteomic comparison based on an intersection protein database from the individual single-species databases. This is less prone to quantification errors arising from differences in the sequences. Consecutively comparing subproteomes of OMV and membranes of the two strains allows distinguishing differences in relative protein amounts caused by global expression changes from those caused by preferential protein packing to OMV or membranes. Among the proteins most differently packed into OMV between the two strains, we detected proteins involved in biosynthesis and metabolism of bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids, as well as some major structural outer membrane proteins. The data are available via ProteomeXchange with identifier PXD022406.
- MeSH
- Francisella tularensis * MeSH
- Francisella MeSH
- lidé MeSH
- proteom genetika MeSH
- proteomika MeSH
- tularemie * MeSH
- virulence MeSH
- vnější bakteriální membrána MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mycobacterium tuberculosis is the main etiological agent of tuberculosis. The Bacillus Calmette-Guérin (BCG) microbes that are primarily used as a vaccine against tuberculosis also constitute the dominant infection model for studying the interaction of mycobacteria with the host cell types. The majority of interaction experiments have been conducted using macrophages and monocytes as prototype phagocyte cell types. Here, we report that M. bovis BCG infects mouse primary B cells as well as human B cell line. The complement receptors, along with B cell receptors, are engaged in the process of bacterial entry into the host B cells. Once inside the B cells, the intracellular trafficking of BCG follows the complete endocytic pathway of the ingested particles, which is in contrast to the events taking place during ingestion of BCG by macrophages. In vivo infection of mice with M. bovis BCG activated peritoneal as well as splenic B cells to produce proinflammatory cytokines. This paper further supports the evidence that B cells are involved in a host's early interactions with intracellular bacterial pathogens and participate in the induction of innate defense responses.
- MeSH
- B-lymfocyty * imunologie mikrobiologie MeSH
- BCG vakcína MeSH
- cytokiny metabolismus MeSH
- lidé MeSH
- Mycobacterium bovis imunologie MeSH
- myši MeSH
- primární buněčná kultura MeSH
- přirozená imunita MeSH
- tuberkulóza imunologie mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
