Hemolytic phospholipaseRv0183of Mycobacterium tuberculosis induces inflammatory response and apoptosis in alveolar macrophage RAW264.7 cells

2010 ◽  
Vol 56 (11) ◽  
pp. 916-924 ◽  
Author(s):  
Guangxian Xu ◽  
Hao Jia ◽  
Yong Li ◽  
Xiaoming Liu ◽  
Min Li ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inflammatory Response ◽  
Alveolar Macrophage ◽  
Cell Apoptosis ◽  
Time Course ◽  
Ectopic Expression ◽  
Host Cells ◽  
Raw264.7 Cells ◽  
Raw264.7 Cell

The metabolic pathway of phospholipids is one of the most important physiologic pathways in Mycobacterium tuberculosis , a typical intracellular bacterium. The hemolytic phospholipase lip gene (Rv0183) is one of 24 phospholipase genes that have been demonstrated to play critical roles in the metabolism of phospholipids in M. tuberculosis. Quantitative RT–PCR and flow cytometry were used to elucidate the immunological and pathogenic implications of the Rv0183 gene on the inflammatory response following persistent expression of Rv0183 in mouse alveolar macrophage RAW264.7 cells. Our results demonstrate that a time-course-dependent ectopic expression of Rv0183 significantly elevated the expression of IL-6, NF-κB, TLR-2, TLR-6, TNFα, and MyD88 in these alveolar macrophage cells. Furthermore, the persistent expression of Rv0183 induced RAW264.7 cell apoptosis in vitro. These findings demonstrate that the expression of Rv0183 induces an inflammatory response and cell apoptosis in the host cells, suggesting that Rv0183 may play an important role in the virulence and pathogenesis of M. tuberculosis infection.

scholarly journals Long non-coding RNA OIP5-AS1 aggravates acute lung injury by promoting inflammation and cell apoptosis via regulating the miR-26a-5p/TLR4 axis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Qingsong Sun ◽  
Man Luo ◽  
Zhiwei Gao ◽  
Xiang Han ◽  
Weiqin Wu ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Inflammatory Response ◽  
Cell Apoptosis ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interacting Protein ◽  
Wide Range

Abstract Background Acute lung injury (ALI) is a pulmonary disorder that leads to acute respiration failure and thereby results in a high mortality worldwide. Increasing studies have indicated that toll-like receptor 4 (TLR4) is a promoter in ALI, and we aimed to explore the underlying upstream mechanism of TLR4 in ALI. Methods We used lipopolysaccharide (LPS) to induce an acute inflammatory response in vitro model and a murine mouse model. A wide range of experiments including reverse transcription quantitative polymerase chain reaction, western blot, enzyme linked immunosorbent assay, flow cytometry, hematoxylin–eosin staining, RNA immunoprecipitation, luciferase activity and caspase-3 activity detection assays were conducted to figure out the expression status, specific role and potential upstream mechanism of TLR4 in ALI. Result TLR4 expression was upregulated in ALI mice and LPS-treated primary bronchial/tracheal epithelial cells. Moreover, miR-26a-5p was confirmed to target TLR4 according to results of luciferase reporter assay. In addition, miR-26a-5p overexpression decreased the contents of proinflammatory factors and inhibited cell apoptosis, while upregulation of TLR4 reversed these effects of miR-26a-5p mimics, implying that miR-26a-5p alleviated ALI by regulating TLR4. Afterwards, OPA interacting protein 5 antisense RNA 1 (OIP5-AS1) was identified to bind with miR-26a-5p. Functionally, OIP5-AS1 upregulation promoted the inflammation and miR-26a-5p overexpression counteracted the influence of OIP5-AS1 upregulation on cell inflammatory response and apoptosis. Conclusion OIP5-AS1 promotes ALI by regulating the miR-26a-5p/TLR4 axis in ALI mice and LPS-treated cells, which indicates a promising insight into diagnostics and therapeutics in ALI.

scholarly journals Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

2016 ◽  
Vol 213 (5) ◽  
pp. 809-825 ◽  
Author(s):  
Yancheng Liu ◽  
Shumin Tan ◽  
Lu Huang ◽  
Robert B. Abramovitch ◽  
Kyle H. Rohde ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Stress Responses ◽  
Drug Action ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Drug Pressure ◽  
Cytokine Activation ◽  
The Impact

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs.

scholarly journals Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Javeed Ahmad ◽  
Aisha Farhana ◽  
Rita Pancsa ◽  
Simran Kaur Arora ◽  
Alagiri Srinivasan ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Terminal Segment ◽  
Host Protein ◽  
Host Cells ◽  
Productive Infection ◽  
Disordered Proteins ◽  
Intrinsically Disordered

ABSTRACT Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis. IMPORTANCE To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells.

scholarly journals Protein Coding and Long Noncoding RNA (lncRNA) Transcriptional Landscape in SARS-CoV-2 Infected Bronchial Epithelial Cells Highlight a Role for Interferon and Inflammatory Response

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 760 ◽  
Author(s):  
Radhakrishnan Vishnubalaji ◽  
Hibah Shaath ◽  
Nehad M. Alajez
Keyword(s):  
Epithelial Cells ◽  
Inflammatory Response ◽  
Noncoding Rna ◽  
Cell Model ◽  
Bronchial Epithelial Cells ◽  
Host Cells ◽  
Protein Coding ◽  
Bronchial Epithelial ◽  
Upstream Regulator

The global spread of COVID-19, caused by pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for an imminent response from medical research communities to better understand this rapidly spreading infection. Employing multiple bioinformatics and computational pipelines on transcriptome data from primary normal human bronchial epithelial cells (NHBE) during SARS-CoV-2 infection revealed activation of several mechanistic networks, including those involved in immunoglobulin G (IgG) and interferon lambda (IFNL) in host cells. Induction of acute inflammatory response and activation of tumor necrosis factor (TNF) was prominent in SARS-CoV-2 infected NHBE cells. Additionally, disease and functional analysis employing ingenuity pathway analysis (IPA) revealed activation of functional categories related to cell death, while those associated with viral infection and replication were suppressed. Several interferon (IFN) responsive gene targets (IRF9, IFIT1, IFIT2, IFIT3, IFITM1, MX1, OAS2, OAS3, IFI44 and IFI44L) were highly upregulated in SARS-CoV-2 infected NBHE cell, implying activation of antiviral IFN innate response. Gene ontology and functional annotation of differently expressed genes in patient lung tissues with COVID-19 revealed activation of antiviral response as the hallmark. Mechanistic network analysis in IPA identified 14 common activated, and 9 common suppressed networks in patient tissue, as well as in the NHBE cell model, suggesting a plausible role for these upstream regulator networks in the pathogenesis of COVID-19. Our data revealed expression of several viral proteins in vitro and in patient-derived tissue, while several host-derived long noncoding RNAs (lncRNAs) were identified. Our data highlights activation of IFN response as the main hallmark associated with SARS-CoV-2 infection in vitro and in human, and identified several differentially expressed lncRNAs during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to SARS-CoV-2 remains to be investigated.

scholarly journals Thyroxine Affects Lipopolysaccharide-Induced Macrophage Differentiation and Myocardial Cell Apoptosis via the NF-κB p65 Pathway Both In Vitro and In Vivo

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Shan Zhu ◽  
Yuan Wang ◽  
Hongtao Liu ◽  
Wen Wei ◽  
Yi Tu ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Cardiac Dysfunction ◽  
Cell Apoptosis ◽  
Cardiac Injury ◽  
Myocardial Cell ◽  
M2 Macrophage ◽  
Mrna Levels ◽  
Cytokine Mrna ◽  
M1 Macrophage

Background. Numerous studies have demonstrated that the inflammatory response is involved in the progression of lipopolysaccharide- (LPS-) induced myocardial cell apoptosis. Accumulating evidence has shown that thyroxine participates in diseases by downregulating the inflammatory response. This study aimed at investigating whether thyroxine alleviates LPS-induced myocardial cell apoptosis. Methods. Bone marrow-derived macrophages (Mø) were treated with LPS and thyroxine, and Mø differentiation and Mø-related cytokine expression were measured. The effect of Mø differentiation on mouse cardiomyocyte (MCM) apoptosis was also detected in vitro. In addition, C57BL/6 mice underwent thyroidectomy and were treated with LPS 35 days later; subsequently, Mø differentiation and myocardial cell apoptosis in hearts were analyzed. To determine whether the nuclear factor-kappa B (NF-κB) p65 pathway mediates the effect of thyroxine on Mø differentiation and myocardial cell apoptosis, the specific NF-κB p65 pathway inhibitor JSH-23 was administered to mice that underwent a thyroidectomy. Results. Levothyroxine treatment significantly reduced the activation of the NF-κB p65 pathway, decreased M1 macrophage (Mø1) differentiation and Mø1-related cytokine mRNA levels in LPS-treated Mø, and increased M2 macrophage (Mø2) differentiation and Mø2-related cytokine mRNA expression. The protective effects of levothyroxine on MCM apoptosis mediated by LPS-treated Mø were alleviated by JSH-23. In mice, thyroidectomy aggravated LPS-induced cardiac injury and cardiac dysfunction, further promoted NF-κB p65 activation, and increased cardiac Mø1 expression and myocardial cell apoptosis but decreased cardiac Mø2 expression. JSH-23 treatment significantly ameliorated the thyroidectomy-induced increases in myocardial cell apoptosis and Mø differentiation. Conclusions. Thyroxine alleviated the Mø1/Mø2 imbalance, reduced the inflammatory response, decreased myocardial cell apoptosis, and protected against cardiac injury and cardiac dysfunction in LPS-treated mice. Thyroxine may be a novel therapeutic strategy to prevent and treat LPS-induced cardiac injury.

An in vitro study of Hoechst 33342 redistribution and its effects on cell viability

2005 ◽  
Vol 24 (11) ◽  
pp. 573-580 ◽  
Author(s):  
N Mohorko ◽  
N Kregar-Velikonja ◽  
G Repovs ◽  
M Gorensek ◽  
M Bresjanac
Keyword(s):  
Cell Death ◽  
Cell Transplantation ◽  
Fluorescence Intensity ◽  
Time Course ◽  
In Vitro Study ◽  
Host Cells ◽  
Hoechst 33342 ◽  
Overnight Incubation ◽  
Donor Cells

Although Hoechst 33342 (H342) is frequently used to label donor cells in cell transplantation research, it has been noted that it might secondarily label the host cells. Furthermore, its potential toxicity leading to cell death has been described. We studied the time course of H342 redistribution from the primary labeled rat bone marrow stromal cells (rBMSC) into the non-labeled rBMSC population over 7 days in culture; we evaluated the nuclear H342 fluorescence intensity as a possible criterion for distinguishing the primary from the secondary labeled cells, and determined the viability of rBMSC after an overnight incubation in 1 mg/mL of H342. H342 labeled / 50% of the initially non-labeled cells within the first 6 hours and almost 90% within a week.Nuclear fluorescence intensity was a reliable criterion for distinguishing primary and secondary labeled cells within the first 24 hours, but less so at later time points. The percentage of either apoptotic or necrotic cells did not rise acutely after the overnight incubation in 1 mg/mL of H342. Although a 12-hour incubation of rBMSC in 1 mg/mL of H342 did not cause acute cell death, H342 rapidly and extensively redistributed into non-labeled cells, which makes H342 a relatively unsuitable marker for cell transplantation research.

Murine Macrophage RAW264.7 Cells Response for the Carbon Nanotubes Immobilized on Substrate

2012 ◽  
Vol 529-530 ◽  
pp. 379-384
Author(s):  
Tsukasa Akasaka ◽  
Shigeaki Abe ◽  
Fumio Watari
Keyword(s):  
Carbon Nanotubes ◽  
Cell Adhesion ◽  
Raw264.7 Cells ◽  
Murine Macrophage ◽  
Proliferation Assay ◽  
Tnf Α ◽  
Raw264.7 Cell ◽  
Cytotoxic Studies ◽  
Cell Adhesion And Proliferation

The reports on cytotoxic studies of carbon nanotubes (CNTs) increased exponentially. In the present study, we investigate murine macrophage RAW264.7 cell response for the CNTs immobilized on a polystyrene substrate. We prepared CNT-coated dishes, and estimate the interaction of RAW264.7 cells with CNTs by cell adhesion, proliferation assay, and measurement of TNF-α production. As a result, the highest cell adhesion and proliferation was observed on a commercially cell culture polystyrene dish, while CNT-coated dish indicate slightly lower activity of them. Moreover, amount of production of TNF-α on the CNT-coated dishes was considerable lower than that in the case of lipopolysaccharide (LPS) addition as a control. These results indicated that CNT-coated dishes could not show strong cytotoxicity for RAW264.7 cellsin vitro.

scholarly journals Contribution of IL-38 in Lung Immunity During Pseudomonas Aeruginosa-Induced Pneumonia

2021 ◽  
Author(s):  
Qiang Wei ◽  
Xi Chen ◽  
Chuanjiang Wang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inflammatory Response ◽  
Murine Model ◽  
Cell Apoptosis ◽  
Protective Role ◽  
Bacterial Removal ◽  
Cytokine Levels ◽  
New Type ◽  
Treg Differentiation

Abstract Objective: Interleukin-38 (IL-38), a new type of cytokine, is involved in processes such as tissue repair, inflammatory response, and immune response. However, its function in pneumonia caused by Pseudomonas aeruginosa is still unclear.Methods: In this study, we detected circulating IL-38 in adults affected by pneumonia caused by P. aeruginosa. The P. aeruginosa-induced pneumonia WT murine model was adopted to evaluate the effect of IL-38 on Treg differentiation, cell apoptosis, survival, tissue damage, inflammation, and bacterial removal.Results: IL-38 is insufficiently secreted in patients who died of P.A. pneumonia.Recombinant IL-38 improved survival, whereas anti-IL-38 antibody reduced survival in the experimental pneumonia murine model. IL-38 exposure reduced the inflammatory response, as suggested by the lung injury, and reduced cytokine levels (IL-1β, IL-6, IL-17A, TNF-α, and CXCL-1, but not IL-10). It also increased bacterial clearance and reduced cell apoptosis in the lungs. Furthermore, IL-38 was shown to reduce TBK1 expression in vitro when naïve CD4+ T lymphocytes were differentiated to Tregs and played a protective role in P.A. pneumonia.Conclusions: To summarize, the above findings provide additional insights into the mechanism of IL-38 in the treatment of P.A. pneumonia.

scholarly journals Integrative Analysis of Human Macrophage Inflammatory Response Related to Mycobacterium tuberculosis Virulence

2021 ◽  
Vol 12 ◽  
Author(s):  
Pauline Bade ◽  
Fabrizio Simonetti ◽  
Stephanie Sans ◽  
Patricia Laboudie ◽  
Khadija Kissane ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inflammatory Response ◽  
Genetic Networks ◽  
Host Cells ◽  
Human Macrophage ◽  
Human Macrophages ◽  
Adaptive Immune ◽  
Adaptive Immune Cells ◽  
Main Host ◽  
Host Inflammatory Response

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, kills 1.5 to 1.7 million people every year. Macrophages are Mtb’s main host cells and their inflammatory response is an essential component of the host defense against Mtb. However, Mtb is able to circumvent the macrophages’ defenses by triggering an inappropriate inflammatory response. The ability of Mtb to hinder phagolysosome maturation and acidification, and to escape the phagosome into the cytosol, is closely linked to its virulence. The modulation of the host inflammatory response relies on Mtb virulence factors, but remains poorly studied. Understanding macrophage interactions with Mtb is crucial to develop strategies to control tuberculosis. The present study aims to determine the inflammatory response transcriptome and miRNome of human macrophages infected with the virulent H37Rv Mtb strain, to identify macrophage genetic networks specifically modulated by Mtb virulence. Using human macrophages infected with two different live strains of mycobacteria (live or heat-inactivated Mtb H37Rv and M. marinum), we quantified and analyzed 184 inflammatory mRNAs and 765 micro(mi)RNAs. Transcripts and miRNAs differently modulated by H37Rv in comparison with the two other conditions were analyzed using in silico approaches. We identified 30 host inflammatory response genes and 37 miRNAs specific for H37Rv virulence, and highlight evidence suggesting that Mtb intracellular-linked virulence depends on the inhibition of IL-1β-dependent pro-inflammatory response, the repression of apoptosis and the delay of the recruitment and activation of adaptive immune cells. Our findings provide new potential targets for the development of macrophage-based therapeutic strategies against TB.

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