scholarly journals Key Macrophage Responses to Infection With Mycobacterium tuberculosis Are Co-Regulated by microRNAs and DNA Methylation

2021 ◽  
Vol 12 ◽  
Author(s):  
Monika Looney ◽  
Rachel Lorenc ◽  
Marc K. Halushka ◽  
Petros C. Karakousis
Keyword(s):  
Dna Methylation ◽  
Mycobacterium Tuberculosis ◽  
Cell Activation ◽  
Immune Cell ◽  
Methylation Status ◽  
Bacterial Pathogen ◽  
Macrophage Infection ◽  
Macrophage Response ◽  
Human Macrophages

Tuberculosis (TB) is the leading cause of death from infection with a single bacterial pathogen. Host macrophages are the primary cell type infected with Mycobacterium tuberculosis (Mtb), the organism that causes TB. Macrophage response pathways are regulated by various factors, including microRNAs (miRNAs) and epigenetic changes that can shape the outcome of infection. Although dysregulation of both miRNAs and DNA methylation have been studied in the context of Mtb infection, studies have not yet investigated how these two processes may jointly co-regulate critical anti-TB pathways in primary human macrophages. In the current study, we integrated genome-wide analyses of miRNA abundance and DNA methylation status with mRNA transcriptomics in Mtb-infected primary human macrophages to decipher which macrophage functions may be subject to control by these two types of regulation. Using in vitro macrophage infection models and next generation sequencing, we found that miRNAs and methylation changes co-regulate important macrophage response processes, including immune cell activation, macrophage metabolism, and AMPK pathway signaling.

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

scholarly journals Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pierre Santucci ◽  
Daniel J. Greenwood ◽  
Antony Fearns ◽  
Kai Chen ◽  
Haibo Jiang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Combination Chemotherapy ◽  
In Vitro Activity ◽  
In Vivo Efficacy ◽  
Human Macrophages ◽  
Ion Microscopy ◽  
Intracellular Localisation

AbstractTo be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.

Abstract P139: The Relationship Between Tissue Sodium Storage, Immune Cell Activation And Salt-sensitive Hypertension

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Melis Sahinoz ◽  
Fernando Elijovich ◽  
Cheryl L Laffer ◽  
Ashley Pitzer ◽  
Thomas G Stewart ◽  
...  
Keyword(s):  
Cell Activation ◽  
Immune Cell ◽  
Small Sample Size ◽  
Salt Sensitivity ◽  
Small Sample ◽  
Sensitivity Index ◽  
Salt Loading ◽  
Immune Cell Activation ◽  
Salt Depletion

Introduction: Salt Sensitivity (SS) of blood pressure (BP) is an independent predictor of death due to cardiovascular disease, but its pathogenesis is poorly understood. Sodium (Na + ) is stored in the skin and muscle interstitium. This hyperosmolar Na + activates monocytes in vitro via oxidative stress with generation of isolevuglandin (isoLG) protein adducts that are immunogenic and activate the adaptive immune system. Methods: Five subjects with essential hypertension discontinued all anti-hypertensive therapy for two weeks before the study. SS was assessed by an inpatient protocol of salt loading (460 mmoL/24h) and salt depletion (10 mmoL/24h, plus furosemide 40 mg x 3). Muscle and skin Na + contents were measured at baseline (BA) by 23 Sodium magnetic resonance imaging ( 23 NaMRI). Urine and serum electrolytes, glomerular filtration rate and the % CD14 + monocytes containing isoLG adducts using flow cytometry were obtained at BA, after salt-loading (HI) and after salt-depletion (LO). All continuous data are displayed as median (interquartile range). Spearman’s correlation was used to test associations. Results: Median age was 54 years (44-55), 60% of subjects were female, screening systolic BP (SBP) was 140 mmHg (134-148), diastolic BP was 88 mmHg (84-99) and BMI was 35 kg/m 2 (30-39). SBP response to salt-depletion (salt-sensitivity index, SSI) varied from -13.8 to +1.8 mmHg. %isoLG + CD14 + cells were 48 (27-65) at BA, 55 (31-56) at HI, and 70 (33-72) at LO (p=0.594, by the Kruskal-Wallis test). The correlation between SSI and delta (Δ) %isoLG LO minus HI, was 0.86, [95% confidence interval (CI), -0.07-0.99] which may suggest conclusively as we gather more data that the greater the SSI, the larger the decrease in isoLGs by salt depletion. Muscle Na + content correlated with 24h urine Na + (BA) (r=0.90, 95% CI, 0.11-0.99), however, the correlation with BP, SSI or isoLGs was inconclusive, potentially due to the small sample size. Skin Na + content correlated with baseline %CD14IsoLG + (r=0.91; 95% CI, 0.12-0.99). Conclusions: Na + intake is a component of the determinants of muscle Na + . Skin Na + is associated with increased isoLGs in monocytes, a marker of immune cell activation. Variability in ΔCD14isoLG may serve as a biomarker for SS of BP in humans.

scholarly journals Administration of cardiac mesenchymal cells modulates innate immunity in the acute phase of myocardial infarction in mice

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yi Kang ◽  
Marjan Nasr ◽  
Yiru Guo ◽  
Shizuka Uchida ◽  
Tyler Weirick ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Innate Immunity ◽  
Mechanism Of Action ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Mesenchymal Cell ◽  
Cell Types

Abstract Although cardiac mesenchymal cell (CMC) therapy mitigates post-infarct cardiac dysfunction, the underlying mechanisms remain unidentified. It is acknowledged that donor cells are neither appreciably retained nor meaningfully contribute to tissue regeneration—suggesting a paracrine-mediated mechanism of action. As the immune system is inextricably linked to wound healing/remodeling in the ischemically injured heart, the reparative actions of CMCs may be attributed to their immunoregulatory properties. The current study evaluated the consequences of CMC administration on post myocardial infarction (MI) immune responses in vivo and paracrine-mediated immune cell function in vitro. CMC administration preferentially elicited the recruitment of cell types associated with innate immunity (e.g., monocytes/macrophages and neutrophils). CMC paracrine signaling assays revealed enhancement in innate immune cell chemoattraction, survival, and phagocytosis, and diminished pro-inflammatory immune cell activation; data that identifies and catalogues fundamental immunomodulatory properties of CMCs, which have broad implications regarding the mechanism of action of CMCs in cardiac repair.

scholarly journals CMET-30. COMPREHENSIVE METHYLOME ANALYSIS OF EGFR-MUTANT PRIMARY LUNG ADENOCARCINOMA AND MATCHED BRAIN METASTASIS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi58-vi58
Author(s):  
Yasin Mamatjan ◽  
Michael Cabanero ◽  
Jeffrey Zuccato ◽  
Jessica Weiss ◽  
Shirin Karimi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Brain Metastasis ◽  
Lung Adenocarcinoma ◽  
Immune Cell ◽  
Methylation Status ◽  
Cell Infiltration ◽  
Immune Cell Infiltration ◽  
Promoter Regions ◽  
Primary Lung Adenocarcinoma ◽  
Egfr Mutant

Abstract Brain metastasis (BM) in patients with EGFR-mutant lung adenocarcinoma is a major determinant of overall survival. Novel insight into the genetic and epigenetic underpinnings of BM development is lacking. The aim of this study is to compare the methylome of EGFR-mutant primary lung adenocarcinoma (EGFRM-PLA) and matched BM to identify important alterations for the mechanisms of BM. Matched EGFRM-PLA and BM tumors from seven patients were profiled using the Illumina Infinium MethylationEPIC BeadChip array. Unsupervised clustering analyses of the 14 samples showed a similar whole DNA methylation signatures between EGFRM-PLA and BM tumors. Furthermore, PCA plot highlighted that seven matched BM and lung tumor samples were clustered together closely based on matching pairs for the most variable probes (2.5K to 10K). These observations indicate high level of concordance and the same cell of origin. However, these fourteen samples clustered into two groups based on tumor site being lung or brain based on 83K differentially methylated CpG sites. Of the 83K probes, 2.4K were either hypermethylated or hypomethylated in all lung samples. A quarter of these 2.4K probes were located in promoter regions. Specifically, we identified differences in methylation status of EGFR/ALK promoter regions in lung tumors versus BM. CNV analyses showed higher deep deletions of chromosomes and genes in BM compared to EGFRM-PLA. Leukocytes unmethylation for purity (LUMP) scores which indicate immune cell infiltration were similar between lung and BM pairs (Mean LUMP_score=0.64) consistent with high immune cell infiltration. Our results indicated a similar whole DNA methylation signature of EGFRM-PLA and matched BM, while comprehensive analysis identified important differentially methylated probes. Distinct differences in CNV alterations were observed in lung versus brain samples. The BM and EGFRM-PLA showed similar tumor purity and immune cell components. Overall, tumor methylation profiling provides clinically important information regarding biology of BM in EGFRM-PLA.

scholarly journals Identification and Repurposing of Trisubstituted Harmine Derivatives as Novel Inhibitors of Mycobacterium tuberculosis Phosphoserine Phosphatase

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 415 ◽  
Author(s):  
Elise Pierson ◽  
Marie Haufroid ◽  
Tannu Priya Gosain ◽  
Pankaj Chopra ◽  
Ramandeep Singh ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Pathogen ◽  
Multidrug Resistant ◽  
Metabolic Enzyme ◽  
Phosphoserine Phosphatase ◽  
Cellular Assays ◽  
Promising Target ◽  
Global Health Issue ◽  
Serine Pathway

Mycobacterium tuberculosis is still the deadliest bacterial pathogen worldwide and the increasing number of multidrug-resistant tuberculosis cases further complicates this global health issue. M. tuberculosis phosphoserine phosphatase SerB2 is a promising target for drug design. Besides being a key essential metabolic enzyme of the pathogen’s serine pathway, it appears to be involved in immune evasion mechanisms. In this work, a malachite green-based phosphatase assay has been used to screen 122 compounds from an internal chemolibrary. Trisubstituted harmine derivatives were found among the best hits that inhibited SerB2 activity. Synthesis of an original compound helped to discuss a brief structure activity relationship evaluation. Kinetics experiments showed that the most potent derivatives inhibit the phosphatase in a parabolic competitive fashion with apparent inhibition constants ( K i ) values in the micromolar range. Their interaction modes with the enzyme were investigated through induced fit docking experiments, leading to results consistent with the experimental data. Cellular assays showed that the selected compounds also inhibited M. tuberculosis growth in vitro. Those promising results may provide a basis for the development of new antimycobacterial agents targeting SerB2.

scholarly journals Urban airborne particle exposure impairs human lung and blood Mycobacterium tuberculosis immunity

Thorax ◽  
2019 ◽  
Vol 74 (7) ◽  
pp. 675-683 ◽  
Author(s):  
Martha Torres ◽  
Claudia Carranza ◽  
Srijata Sarkar ◽  
Yolanda Gonzalez ◽  
Alvaro Osornio Vargas ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mexico City ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Human Lung ◽  
Constitutive Expression ◽  
In Vitro Exposure ◽  
Ifn Γ

RationaleAssociations between urban (outdoor) airborne particulate matter (PM) exposure and TB and potential biological mechanisms are poorly explored.ObjectivesTo examine whether in vivo exposure to urban outdoor PM in Mexico City and in vitro exposure to urban outdoor PM2.5 (< 2.5 µm median aerodynamic diameter) alters human host immune cell responses to Mycobacterium tuberculosis.MethodsCellular toxicity (flow cytometry, proliferation assay (MTS assay)), M. tuberculosis and PM2.5 phagocytosis (microscopy), cytokine-producing cells (Enzyme-linked immune absorbent spot (ELISPOT)), and signalling pathway markers (western blot) were examined in bronchoalveolar cells (BAC) and peripheral blood mononuclear cells (PBMC) from healthy, non-smoking, residents of Mexico City (n=35; 13 female, 22 male). In vivo-acquired PM burden in alveolar macrophages (AM) was measured by digital image analysis.Measurements and main resultsIn vitro exposure of AM to PM2.5 did not affect M. tuberculosis phagocytosis. High in vivo-acquired AM PM burden reduced constitutive, M. tuberculosis and PM-induced interleukin-1β production in freshly isolated BAC but not in autologous PBMC while it reduced constitutive production of tumour necrosis factor-alpha in both BAC and PBMC. Further, PM burden was positively correlated with constitutive, PM, M. tuberculosis and purified protein derivative (PPD)-induced interferon gamma (IFN-γ) in BAC, and negatively correlated with PPD-induced IFN-γ in PBMC.ConclusionsInhalation exposure to urban air pollution PM impairs important components of the protective human lung and systemic immune response against M. tuberculosis. PM load in AM is correlated with altered M. tuberculosis-induced cytokine production in the lung and systemic compartments. Chronic PM exposure with high constitutive expression of proinflammatory cytokines results in relative cellular unresponsiveness.

scholarly journals IMMU-46. EXAMINATION OF THE EFFECTS OF DEXAMETHASONE ON THE EFFICACY OF IMMUNOTHERAPY IN GLIOMA USING GENE-MEDIATED CYTOTOXIC IMMUNOTHERAPY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi129-vi129
Author(s):  
Marilin Koch ◽  
Mykola Zdioruk ◽  
M Oskar Nowicki ◽  
Estuardo Aguilar ◽  
Laura Aguilar ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Symptomatic Treatment ◽  
Tumor Cell Death ◽  
The Impact

Abstract RATIONALE Dexamethasone is frequently used in symptomatic treatment of glioma patients, although it is known to cause immune suppression. Checkpoint inhibitor immunotherapies have not yet been successful in glioma treatments. Gene-mediated cytotoxic immunotherapy (GMCI) is an immunotherapeutic approach that uses aglatimagene besadenovec with an anti-herpetic prodrug to induce immunogenic tumor cell death and immune cell attraction to the tumor site with potent CD8 T cell activation. GMCI is currently in clinical trials for solid tumors including glioblastoma, where it showed encouraging survival results in a Phase 2 study that did not limit the use of dexamethasone. However, the effects of dexamethasone on its efficacy have not been explored. METHODS We investigated the effects of dexamethasone on GMCI in vitro using cytotoxicity and T-cell-killing assays in glioblastoma cell lines. The impact of dexamethasone in vivo was assessed in an orthotopic syngeneic murine glioblastoma model. RESULTS Cyotoxicity assays showed that Dexamethasone has a slight impact on GMCI in vitro. In contrast, we observed a highly significant effect in T-cell-functional assays in which killing was greatly impaired. Immune cell response assays revealed a reduced T-cell proliferation after co-culture with supernatant from dexamethasone or combination treated glioblastoma cells in contrast to GMCI alone. In a murine model, the combination of GMCI and dexamethasone resulted in a significant reduction in median symptom-free survival (29d) in comparison to GMCI alone (39.5d) (P = 0.0184). CONCLUSION Our data suggest that high doses of dexamethasone may negatively impact the efficacy of immunotherapy for glioma, which may be a consequence of impaired T cell function. These results support the idea that there is a need in identifying possible alternatives to dexamethasone to maximize the effectiveness of immunostimulatory therapies such as GMCI.

scholarly journals Neuraminidase inhibition contributes to influenza A virus neutralization by anti-hemagglutinin stem antibodies

2019 ◽  
Vol 216 (2) ◽  
pp. 304-316 ◽  
Author(s):  
Ivan Kosik ◽  
Davide Angeletti ◽  
James S. Gibbs ◽  
Matthew Angel ◽  
Kazuyo Takeda ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Influenza A ◽  
Cell Activation ◽  
Immune Cell ◽  
Broadly Neutralizing Antibodies ◽  
Neutralization Activity ◽  
Immune Cell Activation ◽  
Influenza Virus Hemagglutinin ◽  
Infected Cells

Broadly neutralizing antibodies (Abs) that bind the influenza virus hemagglutinin (HA) stem may enable universal influenza vaccination. Here, we show that anti-stem Abs sterically inhibit viral neuraminidase (NA) activity against large substrates, with activity inversely proportional to the length of the fibrous NA stalk that supports the enzymatic domain. By modulating NA stalk length in recombinant IAVs, we show that anti-stem Abs inhibit virus release from infected cells by blocking NA, accounting for their in vitro neutralization activity. NA inhibition contributes to anti-stem Ab protection in influenza-infected mice, likely due at least in part to NA-mediated inhibition of FcγR-dependent activation of innate immune cells by Ab bound to virions. Food and Drug Administration–approved NA inhibitors enhance anti-stem–based Fc-dependent immune cell activation, raising the possibility of therapeutic synergy between NA inhibitors and anti-stem mAb treatment in humans.

scholarly journals GABAA receptor α4-subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model

2017 ◽  
Vol 313 (2) ◽  
pp. L406-L415 ◽  
Author(s):  
Gene T. Yocum ◽  
Damian L. Turner ◽  
Jennifer Danielsson ◽  
Matthew B. Barajas ◽  
Yi Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Immune Cells ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Asthma Model

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation ( P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice ( P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration ( P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

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