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Exosomes in serum‑free cultures of THP‑1 macrophages infected with Mycobacterium tuberculosis.

ABSTRACT: It has been shown from the isolation and characterization of exosomes from cell culture media supplemented with fetal bovine serum that both their quality and purity are affected. The high abundance of serum proteins, including bovine cell derived exosomes, is also a potential source of contaminants, which may result in appreciable yields of impure exosomes, thereby leading to artifacts. Isolation and characterization of exosomes from cells maintained under serum‑free conditions should therefore ensure the high quality necessary for medical applications. To meet this end, the present study aimed to characterize exosomes released from THP‑1 macrophages cultured in serum‑free, ultra‑centrifuged medium upon infection with the human pathogen Mycobacterium tuberculosis (Mtb). Macrophages differentiated from the human cell line THP‑1 were infected at a multiplicity of infection (MOI) of 5. Macrophages were cultivated in CellGenix^® GMP DC serum‑free ultra‑centrifuged medium for 4, 24 and 48 h at 37˚C in a humidified atmosphere with 5% CO₂. Total exosome isolation reagent was used to extract the exosomes from the cell culture supernatants of naïve and Mtb‑infected THP‑1 macrophages. The size and purity of the exosomes isolated were subsequently assessed by various methods, including nanoparticle tracking analysis, flow cytometry, MACSPlex exosome analysis, and western blotting. The serum‑free, ultra‑centrifuged medium was found to support the proliferation of the THP‑1 cells successfully. The nanoparticle tracking analysis data revealed that the majority of the isolated particles were within the size range of exosomes (i.e., 30‑150 nM). The MACSPlex exosome analysis confirmed the expression of the exosomal markers, CD9, CD63 and CD81. Furthermore, western blot analysis of the isolated exosomes indicated the presence of CD9, CD63, CD81 and lysosomal associated membrane protein‑1 (LAMP‑1), and also confirmed the absence of Mtb proteins. Taken together, these data provide evidence that serum‑free, ultra‑centrifuged CellGenix^® GMP DC medium is suitable for application in exosome research, and may significantly advance such studies. Therefore, the use of serum‑free medium for exosome isolation purposes could offer considerable advantages, and constitute a significant improvement in the growing field of extracellular vesicle research. The use of more sensitive methods represents an advance that will enable researchers to rule out the presence of Mtb pathogenic proteins in exosomes isolated from infected serum‑free cell cultures.

SUBMITTER: Biadglegne F

PROVIDER: S-EPMC8477185 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tuberculosis (TB) remains a worldwide healthcare concern, and the exploration of the host-pathogen interaction is essential to develop therapeutic modalities and strategies to control Mycobacterium tuberculosis (M.tb). In this study, RNA sequencing (transcriptome sequencing) was employed to investigate the global transcriptome changes in the macrophages during the different strains of M.tb infection. THP-1 cells derived from macrophages were exposed to the virulent M.tb strain H37Rv (Rv) or the avirulent M.tb strain H37Ra (Ra), and the M.tb BCG vaccine strain was used as a control. The cDNA libraries were prepared from M.tb-infected macrophages and then sequenced. To assess the transcriptional differences between the expressed genes, the bioinformatics analysis was performed using a standard pipeline of quality control, reference mapping, differential expression analysis, protein-protein interaction (PPI) networks, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Q-PCR and Western blot assays were also performed to validate the data. Our findings indicated that, when compared to BCG or M.tb H37Ra infection, the transcriptome analysis identified 66 differentially expressed genes in the M.tb H37Rv-infected macrophages, out of which 36 genes were up-regulated, and 30 genes were down-regulated. The up-regulated genes were associated with immune response regulation, chemokine secretion, and leucocyte chemotaxis. In contrast, the down-regulated genes were associated with amino acid biosynthetic and energy metabolism, connective tissue development and extracellular matrix organization. The Q-PCR and Western blot assays confirmed increased expression of pro-inflammatory factors, altered energy metabolic processes, enhanced activation of pro-inflammatory signalling pathways and increased pyroptosis in H37Rv-infected macrophage. Overall, our RNA sequencing-based transcriptome study successfully identified a comprehensive, in-depth gene expression/regulation profile in M.tb-infected macrophages. The results demonstrated that virulent M.tb strain H37Rv infection triggers a more severe inflammatory immune response associated with increased tissue damage, which helps in understanding the host-pathogen interaction dynamics and pathogenesis features in different strains of M.tb infection.

Project description:Mycobacterium tuberculosis (M. tb) is a highly successful pathogen that has co?existed with humans for 1,000's of years. As the cornerstone of the immune system, macrophages are a key part of innate immunity. They ingest and degrade foreign substances including aging cells and microorganisms, coordinate the inflammatory process, and are the first line of defense against M. tb infection. Recent advances in cellular mycobacteriology have indicated that M. tb uses an remarkably complex strategy to disrupt macrophage function, in order to counteract the antimicrobial mechanisms of the innate and adaptive immune responses, thereby achieving immune escape. With the popularity of microarray technology, a variety of public platforms have provided a variety of gene expression data associated with physiological and disease conditions. Meta?analysis can systematically and quantitatively analyze multiple independent data concerning the same disease, greatly improving the statistical significance and credibility of the gene expression data analysis performed. In the present study, 6 microarray expression datasets of human acute monocytic leukemia THP?1 cell line infected by M. tb H37Rv strain were collected from the GEO database. A total of 4 high?quality datasets were identified using meta?analysis methods in R language, and 306 differentially expressed genes with statistical significance were obtained. Then, a protein?protein interaction (PPI) network of these differentially expressed genes was constructed on the Search Tool for the Retrieval of Interacting Genes/Proteins Database online tool and visualized by Cytoscape v. 3.6.1 software. Using CentiScape and MCODE plugin in the Cytoscape software to mine the functional modules associated with M. tb infection process, 32 characteristic genes were identified. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis was performed on the 32 characteristic genes, and it was demonstrated that these genes were primarily associated with the type I interferon (IFN) pathway. In the established model of THP?1?derived macrophages infected by M. tb, the actual differential expression levels of IFN?stimulated gene 15 (ISG15), 2'?5?oligoadenylate synthetase like (OASL), IFN regulatory factor 7 (IRF7) and DExD/H?box helicase 58 (DDX58), the first 4 genes of the 32 characteristic genes, were verified by reverse transcription quantitative polymerase chain reaction. The results were consistent with the results of microarray analysis. The association between ISG15, OASL and IRF7 and TB infection was also verified. Although a number of studies have identified that the type I IFN pathway may assist M. tb to achieve immune escape, the present study used a meta?analysis of microarray data and PPI network analysis to examine some of the novel genes identified in the IFN pathway. The results furthered the understanding of the molecular mechanisms of the TB immune response and provided a novel perspective for future therapeutic goals.

Project description:BackgroundTuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb) remains a significant health problem worldwide with a third of the world population infected and nearly nine million new cases claiming 1.1 million deaths every year. The outcome following infection by Mtb is determined by a complex and dynamic host-pathogen interaction in which the phenotype of the pathogen and the immune status of the host play a role. However, the molecular mechanism by which Mtb strains induce different responses during intracellular infection of the host macrophage is not fully understood. To explore the early molecular events triggered upon Mtb infection of macrophages, we studied the transcriptional responses of murine bone marrow-derived macrophages (BMM) to infection with two clinical Mtb strains, CDC1551 and HN878. These strains have previously been shown to differ in their virulence/immunogenicity in the mouse and rabbit models of pulmonary TB.ResultsIn spite of similar intracellular growth rates, we observed that compared to HN878, infection by CDC1551 of BMM was associated with an increased global transcriptome, up-regulation of a specific early (6 hours) immune response network and significantly elevated nitric oxide production. In contrast, at 24 hours post-infection of BMM by HN878, more host genes involved in lipid metabolism, including cholesterol metabolism and prostaglandin synthesis were up-regulated, compared to infection with CDC1551.In association with the differences in the macrophage responses to infection with the 2 Mtb strains, intracellular CDC1551 expressed higher levels of stress response genes than did HN878.ConclusionsIn association with the early and more robust macrophage activation, intracellular CDC1551 cells were exposed to a higher level of stress leading to increased up-regulation of the bacterial stress response genes. In contrast, sub-optimal activation of macrophages and induction of a dysregulated host cell lipid metabolism favored a less stressful intracellular environment for HN878. Our findings suggest that the ability of CDC1551 and HN878 to differentially activate macrophages during infection probably determines their ability to either resist host cell immunity and progress to active disease or to succumb to the host protective responses and be driven into a non-replicating latent state in rabbit lungs.

Project description:A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.

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Exosomes in serum‑free cultures of THP‑1 macrophages infected with Mycobacterium tuberculosis.

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