Mycobacterium tuberculosis infection up-regulates MFN2 expression to promote NLRP3 inflammasome formation

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide, especially in children. However, the mechanisms by which MTB infects its cellular host, activates an immune response, and triggers inflammation remain unknown. Mitochondria play important roles in the initiation and activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, where mitochondria-associated endoplasmic reticulum membranes (MAMs) may serve as the platform for inflammasome assembly and activation. Additionally, mitofusin 2 (MFN2) is implicated in the formation of MAMs, but, the roles of mitochondria and MFN2 in MTB infection have not been elucidated. Using mircroarry profiling of TB patients and in vitro MTB stimulation of macrophages, we observed an up-regulation of MFN2 in the peripheral blood mononuclear cells of active TB patients. Furthermore, we found that MTB stimulation by MTB-specific antigen ESAT-6 or lysate of MTB promoted MFN2 interaction with NLRP3 inflammasomes, resulting in the assembly and activation of the inflammasome and, subsequently, IL-1β secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection.

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Simvastatin Suppresses Interleukin Iβ Release in Human Peripheral Blood Mononuclear Cells Stimulated With Cholesterol Crystals

Journal of Cardiovascular Pharmacology and Therapeutics ◽

10.1177/1074248418776261 ◽

2018 ◽

Vol 23 (6) ◽

pp. 509-517 ◽

Cited By ~ 10

Author(s):

Anna J. Boland ◽

Nisha Gangadharan ◽

Pierce Kavanagh ◽

Linda Hemeryck ◽

Jennifer Kieran ◽

...

Keyword(s):

Cardiovascular Disease ◽

Peripheral Blood Mononuclear Cells ◽

Nlrp3 Inflammasome ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

Statins are mainstream therapy in the treatment and prevention of cardiovascular disease through inhibitory effects on cholesterol synthesis. However, statins’ beneficial effects in cardiovascular disease may also be attributable to their role as anti-inflammatory mediators. Here, we investigated the effects of simvastatin treatment on expression levels of interleukin (IL) 1β in both patient with hyperlipidemia and healthy human peripheral blood mononuclear cells (PBMCs) using cholesterol crystals (CC), a cardiovascular pathogenic stimulus for activation of the NOD-like receptor pyrin domain–containing protein 3 (NLRP3) inflammasome. Cholesterol crystal-induced NLRP3 inflammasome activation was used to trigger maturation and release of IL-1β in PBMCs. Specifically, isolated PBMCs from patients with hyperlipidemia at baseline and following 8 weeks of in vivo treatment with simvastatin (10-20 mg) daily were stimulated with lipopolysaccharide (LPS; 100 ng/mL) for 3 hours to induce proIL-Iβ expression followed by CC (2 mg/mL) stimulation for further 18 hours to activate the NLRP3 inflammasome complex to induce maturation/activation of IL-1β. Peripheral blood mononuclear cells were also isolated from healthy donors and stimulated in vitro with simvastatin (50, 25, 5, and 2 µmol/L) prior to stimulation with LPS and CC as described above. The effects of simvastatin treatment on levels of IL-1β expression were determined by enzyme-linked immunosorbent assay and western blot. Both in vitro and in vivo treatments with simvastatin led to a significant reduction in the levels of expression of IL-1β in response to stimulation with CC. Simvastatin inhibits the expression and activation of IL-1β induced by CC in PBMCs, which may contribute to its protective role in patients with cardiovascular disease.

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Autoinflammatory disease with corneal and mucosal dyskeratosis caused by a novel NLRP1 variant

Rheumatology ◽

10.1093/rheumatology/kez612 ◽

2019 ◽

Vol 59 (9) ◽

pp. 2334-2339 ◽

Cited By ~ 3

Author(s):

Troels Herlin ◽

Sofie E Jørgensen ◽

Christian Høst ◽

Patrick S Mitchell ◽

Maria Hønholt Christensen ◽

...

Keyword(s):

Sanger Sequencing ◽

Mononuclear Cells ◽

Novel Mutation ◽

Mucosal Inflammation ◽

Inflammasome Activation ◽

Incomplete Penetrance ◽

Peripheral Blood Mononuclear ◽

Pyrin Domain ◽

Patient Pbmcs

Abstract Objectives Here we investigated a patient with inflammatory corneal intraepithelial dyskeratosis, mucosal inflammation, tooth abnormalities and, eczema to uncover the genetic and immunological basis of the disease. Methods On suspicion of an autoinflammatory condition, Sanger sequencing of nucleotide-binding oligomerization domain-like, leucine-rich repeat pyrin domain containing 1 (NLRP1) was performed and combined with an in vitro inflammasome reconstitution assay to measure caspase-1-mediated IL-1β cleavage, stimulation of patient peripheral blood mononuclear cells (PBMCs) and whole blood to measure IL-1β, IL-18 production and quantification of apoptosis-associated speck-like protein containing CARD (ASC) speck formation as a measure of inflammasome activation by flow cytometry. Results Sanger sequencing revealed a novel mutation (c.175G>C, p.A59P; NM_33004.4) in the inflammasome molecule NLRP1 segregating with disease, although with incomplete penetrance, in three generations. We found that patient PBMCs produced increased IL-1β in response to inflammatory stimuli, as well as increased constitutive levels of IL-18. Moreover, we demonstrate that expression of the identified NLRP1 A59P variant caused spontaneous IL-1β cleavage to mature IL-1β. In addition, patient PBMCs responded to NLRP1 stimulation with increased ASC speck formation as a reflection of elevated inflammasome activity. Conclusion We demonstrate that this novel NLRP1 A59P variant caused increased activation of the NLRP1 inflammasome, resulting in constitutively and inducibly elevated IL-1β and IL-18 synthesis. We suggest the NLRP1 mutation underlies the pathogenesis of this rare autoinflammatory dyskeratotic disease inherited in an autosomal dominant manner with incomplete penetrance in the patient and within the family for several generations.

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Applications of Transcriptomics and Proteomics for Understanding Dormancy and Resuscitation in Mycobacterium tuberculosis

Frontiers in Microbiology ◽

10.3389/fmicb.2021.642487 ◽

2021 ◽

Vol 12 ◽

Author(s):

Manikuntala Kundu ◽

Joyoti Basu

Keyword(s):

Mycobacterium Tuberculosis ◽

Fatty Acid ◽

Cell Division ◽

Drug Targets ◽

Mononuclear Cells ◽

Clinical Symptoms ◽

Transcriptional Regulator ◽

Peripheral Blood Mononuclear ◽

Latently Infected

Mycobacterium tuberculosis can survive within its host for extended periods of time without any clinical symptoms of disease and reactivate when the immune system is weakened. A detailed understanding of how M. tuberculosis enters into and exits out of dormancy, is necessary in order to develop new strategies for tackling tuberculosis. Omics methodologies are unsupervised and unbiased to any hypothesis, making them useful tools for the discovery of new drug targets. This review summarizes the findings of transcriptomic and proteomic approaches toward understanding dormancy and reactivation of M. tuberculosis. Within the granuloma of latently infected individuals, the bacteria are dormant, with a marked slowdown of growth, division and metabolism. In vitro models have attempted to simulate these features by subjecting the bacterium to hypoxia, nutrient starvation, potassium depletion, growth in the presence of vitamin C, or growth in the presence of long-chain fatty acids. The striking feature of all the models is the upregulation of the DosR regulon, which includes the transcriptional regulator Rv0081, one of the central hubs of dormancy. Also upregulated are chaperone proteins, fatty acid and cholesterol degrading enzymes, the sigma factors SigE and SigB, enzymes of the glyoxylate and the methylcitrate cycle, the Clp proteases and the transcriptional regulator ClgR. Further, there is increased expression of genes involved in mycobactin synthesis, fatty acid degradation, the glyoxylate shunt and gluconeogenesis, in granulomas formed in vitro from peripheral blood mononuclear cells from latently infected individuals compared to naïve individuals. Genes linked to aerobic respiration, replication, transcription, translation and cell division, are downregulated during dormancy in vitro, but upregulated during reactivation. Resuscitation in vitro is associated with upregulation of genes linked to the synthesis of mycolic acids, phthiocerol mycocerosate (PDIM) and sulfolipids; ribosome biosynthesis, replication, transcription and translation, cell division, and genes encoding the five resuscitation promoting factors (Rpfs). The expression of proteases, transposases and insertion sequences, suggests genome reorganization during reactivation.

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Increase of CD4+CD25highFoxP3+ cells impairs in vitro human microbicidal activity against Mycobacterium tuberculosis during latent and acute pulmonary tuberculosis

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009605 ◽

2021 ◽

Vol 15 (7) ◽

pp. e0009605

Author(s):

Lorenzzo Lyrio Stringari ◽

Luciana Polaco Covre ◽

Flávia Dias Coelho da Silva ◽

Vivian Leite de Oliveira ◽

Maria Carolina Campana ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

Mononuclear Cells ◽

Critical Role ◽

Latent Tuberculosis ◽

Treg Cells ◽

Host Responses ◽

Peripheral Blood Mononuclear ◽

Microbicidal Activity

Background Regulatory T cells (Tregs) play a critical role during Mycobacterium tuberculosis (Mtb) infection, modulating host responses while neutralizing excessive inflammation. However, their impact on regulating host protective immunity is not completely understood. Here, we demonstrate that Treg cells abrogate the in vitro microbicidal activity against Mtb. Methods We evaluated the in vitro microbicidal activity of peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis (TB), individuals with latent tuberculosis infection (LTBI, TST+/IGRA+) and healthy control (HC, TST-/IGRA-) volunteers. PBMCs, depleted or not of CD4+CD25+ T-cells, were analyzed to determine frequency and influence on microbicidal activity during in vitro Mtb infection with four clinical isolates (S1, S5, R3, and R6) and one reference strain (H37Rv). Results The frequency of CD4+CD25highFoxP3+ cells were significantly higher in Mtb infected whole blood cultures from both TB patients and LTBI individuals when compared to HC. Data from CD4+CD25+ T-cells depletion demonstrate that increase of CD4+CD25highFoxP3+ is associated with an impairment of Th-1 responses and a diminished in vitro microbicidal activity of LTBI and TB groups. Conclusions Tregs restrict host anti-mycobacterial immunity during active disease and latent infection and thereby may contribute to both disease progression and pathogen persistence.

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Tuberculostearic acid (TSA)-containing phosphatidylinositols as reliable marker to determine Mycobacterium tuberculosis bacterial burden

10.1101/2021.02.04.429149 ◽

2021 ◽

Author(s):

Julius Brandenburg ◽

Jan Heyckendorf ◽

Franziska Waldow ◽

Nicole Zehethofer ◽

Lara Linnemann ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mononuclear Cells ◽

Model Systems ◽

Human Neutrophils ◽

Bacterial Burden ◽

Peripheral Blood Mononuclear ◽

Causative Agents ◽

Tuberculostearic Acid ◽

Experimental Model Systems

AbstractIt is estimated that approximately one-fourth of the world's population is infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agents of tuberculosis (TB). In this study, we present rationally developed molecular markers for bacterial burden, which are derived from mycobacterial phospholipids. Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PI) are present in all clinically relevant MTBC lineages investigated. For the major abundant lipid PI 16:0_19:0 (TSA), a detection limit equivalent to 102 colony forming units (CFU) was determined for bacterial cultures and approximately 103 for cell culture systems. We further developed a mass spectrometry based targeted lipid assay, which – in contrast to bacterial quantification on solid medium – can be performed within several hours including sample preparation. Translation of this indirect and culture-free detection approach allowed the determination of pathogen loads in infected murine macrophages, human neutrophils and murine lung tissue. We show that marker lipids inferred from the mycobacterial PIs are increased in peripheral blood mononuclear cells (PBMCs) of TB patients beyond the lipid metabolic background in comparison to healthy controls. In a small cohort of drug-susceptible TB patients elevated levels of these marker molecules were detected at therapy start and declined following successful anti-tuberculosis treatment. The concentration of TSA-containing PIs can be used as correlate for reliable and rapid quantification of Mycobacterium tuberculosis (Mtb) burden in experimental in vitro model systems and may also provide a clinically relevant tool for monitoring TB therapy.One Sentence SummaryTuberculostearic acid containing phosphatidylinositols represent a novel, fast to measure, reliable correlate of Mycobacterium tuberculosis bacterial burden in experimental model systems, which makes a future clinical application conceivable.

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Regulation of Interferon-γ receptor (IFN-γR) expression in macrophages during Mycobacterium tuberculosis infection

BioMolecular Concepts ◽

10.1515/bmc-2020-0006 ◽

2020 ◽

Vol 11 (1) ◽

pp. 76-85

Author(s):

Gunjan Kak ◽

Brijendra K Tiwari ◽

Yogendra Singh ◽

Krishnamurthy Natarajan

Keyword(s):

Mycobacterium Tuberculosis ◽

Mononuclear Cells ◽

Second Messengers ◽

Surface Expression ◽

Interferon Γ ◽

Fine Tuning ◽

Mycobacterium Tuberculosis Infection ◽

Peripheral Blood Mononuclear ◽

Negative Role ◽

Ifn Γ

AbstractInterferon-gamma (IFN-γ) is a key cytokine that mediates immunity to tuberculosis (TB). Mycobacterium tuberculosis (M. tb) is known to downregulate the surface expression of IFN-γ receptor (IFN-γR) on macrophages and peripheral blood mononuclear cells (PBMCs) of patients with active TB disease. Many M. tb antigens also downmodulate IFN-γR levels in macrophages when compared with healthy controls. In the current study, we aimed at deciphering key factors involved in M. tb mediated downregulation of IFN-γR levels on macrophage surface. Our data showed that both M. tb H37Rv and M. bovis BCG infections mediate downmodulation of IFN-γR on human macrophages. This downmodulation is regulated at the level of TLR signaling pathway, second messengers such as calcium and cellular kinases i.e. PKC and ERK-MAPK, indicating that fine tuning of calcium response is critical to maintaining IFN-γR levels on macrophage surface. In addition, genes in the calcium and cysteine protease pathways which were previously identified by us to play a negative role during M. tb infection, also regulated IFN-γR expression. Thus, modulations in IFN-γR levels by utilizing host machinery may be a key immune suppressive strategy adopted by the TB pathogen to ensure its persistence and thwart host defense.

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SFTSV Infection Induced Interleukin-1β Secretion Through NLRP3 Inflammasome Activation

Frontiers in Immunology ◽

10.3389/fimmu.2021.595140 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jian-Wei Liu ◽

Min Chu ◽

Yong-jun Jiao ◽

Chuan-Min Zhou ◽

Rui Qi ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Hemorrhagic Fever ◽

Interleukin 1Β ◽

Inflammasome Activation ◽

Peripheral Blood Mononuclear ◽

Nlrp3 Inflammasome Activation ◽

Novel Drugs ◽

Domain 3

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus that causes hemorrhagic fever. Previous studies showed that SFTSV-infected patients exhibited elevated levels of pro-inflammatory cytokines like interleukin-1β (IL-1β), indicating that SFTSV infection may activate inflammasomes. However, the detailed mechanism remains poorly understood. Herein, we found that SFTSV could stimulate the IL-1β secretion in the infected human peripheral blood mononuclear cells (PBMCs), human macrophages, and C57/BL6 mice. We demonstrate that the maturation and secretion of IL-1β during SFTSV infection is mediated by the nucleotide and oligomerization domain, leucine-rich repeat-containing protein family, pyrin-containing domain 3 (NLRP3) inflammasome. This process is dependent on protease caspase-1, a component of the NLRP3 inflammasome complex. For the first time, our study discovered the role of NLRP3 in response to SFTSV infection. This finding may lead to the development of novel drugs to impede the pathogenesis of SFTSV infection.

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Precipitation of Soluble Uric Acid Is Necessary for In Vitro Activation of the NLRP3 Inflammasome in Primary Human Monocytes

The Journal of Rheumatology ◽

10.3899/jrheum.180855 ◽

2019 ◽

Vol 46 (9) ◽

pp. 1141-1150 ◽

Cited By ~ 7

Author(s):

Ben M. Alberts ◽

James S. Barber ◽

Sandra M. Sacre ◽

Kevin A. Davies ◽

Pietro Ghezzi ◽

...

Keyword(s):

Uric Acid ◽

Nlrp3 Inflammasome ◽

Mononuclear Cells ◽

Prolonged Exposure ◽

Human Peripheral Blood ◽

Elevated Serum ◽

Human Monocytes ◽

Peripheral Blood Mononuclear ◽

Msu Crystals

Objective.To investigate the effects of soluble uric acid (UA) on expression and activation of the NOD-like receptor (NLR) pyrin domain containing protein 3 (NLRP3) inflammasome in human monocytes to elucidate the role of hyperuricemia in the pathogenesis of gout.Methods.Primary human monocytes and the THP-1 human monocyte cell line were used to determine the effects of short- and longterm exposure to UA on activation of the NLRP3 inflammasome and subsequent interleukin 1β (IL-1β) secretion by ELISA and cell-based assays. Expression of key NLRP3 components in monocytes from patients with a history of gout were analyzed by quantitative PCR.Results.Precipitation of UA was required for activation of the NLRP3 inflammasome and subsequent release of IL-1β in human monocytes. Neither monosodium urate (MSU) crystals nor soluble UA had any effect on activation of the transcription factor, nuclear factor-κB. Prolonged exposure of monocytes to soluble UA did not alter these responses. However, both MSU crystals and soluble UA did result in a 2-fold increase in reactive oxygen species. Patients with gout (n = 15) had significantly elevated serum UA concentrations compared to healthy individuals (n = 16), yet secretion of IL-1β and expression of NLRP3 inflammasome components in monocytes isolated from these patients were not different from those of healthy controls.Conclusion.Despite reports indicating that soluble UA can prime and activate the NLRP3 inflammasome in human peripheral blood mononuclear cells, precipitation of soluble UA into MSU crystals is essential for in vitro NLRP3 signaling in primary human monocytes.

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Modulation of Gamma Interferon Receptor 1 by Mycobacterium tuberculosis: a Potential Immune Response Evasive Mechanism

Infection and Immunity ◽

10.1128/iai.01743-06 ◽

2007 ◽

Vol 75 (5) ◽

pp. 2500-2510 ◽

Cited By ~ 36

Author(s):

Amit Singhal ◽

Anand Jaiswal ◽

Virendra K. Arora ◽

Hanumanthappa K. Prasad

Keyword(s):

Mycobacterium Tuberculosis ◽

Mrna Expression ◽

Mononuclear Cells ◽

Surface Expression ◽

Gamma Interferon ◽

Mobility Shift ◽

Down Regulation ◽

Peripheral Blood Mononuclear ◽

Ifn Γ

ABSTRACT Mycobacterium tuberculosis inhibits gamma interferon (IFN-γ)-mediated antimycobacterial action by adopting diverse mechanisms. IFN-γ binds to its receptor, IFN-γR, in order to initiate proper signaling. We have observed reduced surface expression levels of IFN-γ receptor 1 (IFN-γR1) in untreated pulmonary tuberculosis patients compared to those in healthy individuals (P < 0.01). Following antitubercular therapy, the expression of IFN-γR1 was restored in these patients. To delineate the mechanism by which M. tuberculosis modulates IFN-γR1, in vitro experiments were designed, wherein the down modulation of IFN-γR1 surface expression was observed for CD14+ cells in peripheral blood mononuclear cells (PBMCs) cocultured with live M. tuberculosis compared to that for uninfected cells (P < 0.01). No modulation of IFN-γR1 expression was observed for CD14+ cells in PBMCs infected with Mycobacterium smegmatis. A time-dependent decrease in IFN-γR1 mRNA expression was observed for PBMCs infected with M. tuberculosis. Similar down modulation of IFN-γR1 protein and mRNA expression in phorbol myristate acetate-differentiated THP-1 cells (pdTHP-1) by M. tuberculosis was observed (P < 0.01). Using reporter gene analysis of 5′ deletion constructs of the IFN-γR1 gene (IFNGR1) promoter, the decrease in IFN-γR1 mRNA in M. tuberculosis-infected pdTHP-1 cells was shown to be due to the decreased transcription of IFNGR1. By immunoblotting and electrophoretic mobility shift assays, the down regulation of stimulating protein 1 (Sp1) expression and its recruitment on the phorbol ester-responsive element of the IFNGR1 promoter in M. tuberculosis-infected pdTHP-1 cells was observed. This down regulation of Sp1 in pdTHP-1 cells cocultured with M. tuberculosis may be responsible for the down regulation of IFN-γR1 expression, thereby potentially altering its receptivity to IFN-γ.

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Characterizing the NLRP3 Inflammasome in Mood Disorders: Overview, Technical Development, and Measures of Peripheral Activation in Adolescent Patients

International Journal of Molecular Sciences ◽

10.3390/ijms222212513 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12513

Author(s):

Xinyang Y. Zhou ◽

Shehan M. Fernando ◽

Alexander Y. Pan ◽

Rebecca Laposa ◽

Kathryn R. Cullen ◽

...

Keyword(s):

Mood Disorders ◽

Nlrp3 Inflammasome ◽

Depressive Disorders ◽

Mononuclear Cells ◽

Interleukin 1 ◽

Measurement Techniques ◽

Mrna Levels ◽

Peripheral Blood Mononuclear ◽

Pyrin Domain ◽

Peripheral Activation

The NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome is a node of intracellular stress pathways and a druggable target which integrates mitochondrial stress and inflammatory cascades. While a body of evidence suggests the involvement of the NLRP3 inflammasome in numerous diseases, a lack of reliable measurement techniques highlights the need for a robust assay using small quantities of biological samples. We present a literature overview on peripheral activation of the NLRP3 inflammasome in mood disorders, then outline a process to develop and validate a robust assay to measure baseline and activated intracellular levels of “apoptosis-associated speck-like protein containing a CARD” (ASC) as a key component of an inflammatory profile in peripheral blood mononuclear cells (PBMC). A consistent association between high NLRP3 mRNA levels and relevant cytokines was seen in the literature. Using our method to measure ASC, stimulation of PBMC with lipopolysaccharide and nigericin or adenosine triphosphate resulted in microscopic identification of intracellular ASC specks, as well as interleukin 1 (IL-1) beta and caspase-1 p10 in the periphery. This was abolished by dose-dependent pre-treatment with 100 nM MCC950. We also report the use of this technique in a small pilot sample from patients with bipolar disorder and depressive disorders. The results show that levels of intracellular ASC and IL-1 beta are sensitive to change upon activation and maintained over time, which may be used to improve the detection of NLRP3 activation and guide personalized therapeutic strategy in the treatment of patients.

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