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

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.

Elevated Expression of the NLRP3 Inflammasome and Its Correlation with Disease Activity in Adult-onset Still Disease

2017 ◽  
Vol 44 (8) ◽  
pp. 1142-1150 ◽  
Author(s):  
Chia-Wei Hsieh ◽  
Yi-Ming Chen ◽  
Chi-Chen Lin ◽  
Kuo-Tung Tang ◽  
Hsin-Hua Chen ◽  
...  
Keyword(s):  
Disease Activity ◽  
Mrna Expression ◽  
Nlrp3 Inflammasome ◽  
Mononuclear Cells ◽  
Mrna Levels ◽  
Adult Onset ◽  
Peripheral Blood Mononuclear ◽  
Protein Expressions ◽  
Still Disease ◽  
Stimulation Of

Objective.The dysregulation of the NLRP3 (NLR containing a pyrin domain) inflammasome is involved in autoinflammatory diseases. Adult-onset Still disease (AOSD) is regarded as an autoinflammatory disease. However, the pathogenic involvement of NLRP3 inflammasome in AOSD remains unclear and NLRP3 activators in AOSD are currently unknown.Methods.The mRNA expression of NLRP3 inflammasome signaling in peripheral blood mononuclear cells (PBMC) from 34 patients with AOSD and 14 healthy subjects was determined using quantitative-PCR (qPCR). The changes in mRNA and protein levels of NLRP3 inflammasome signaling in PBMC treated with the potential activator [imiquimod (IMQ)] or inhibitor of NLRP3 were evaluated using qPCR and immunoblotting, respectively. The supernatant levels of interleukin (IL)-1β and IL-18 were determined by ELISA.Results.Significantly higher mRNA levels of NLRP3 inflammasome signaling were observed in patients with AOSD compared with healthy controls. NLRP3 expressions were positively correlated with disease activity in patients with AOSD. IMQ (an effective Toll-like receptor 7 ligand; 10 µg/ml and 25 µg/ml) stimulation of PBMC from patients with AOSD induced dose-dependent increases of mRNA expression of NLRP3 (mean ± standard error of the mean, 2.06 ± 0.46 and 6.05 ± 1.84, respectively), caspase-1 (1.81 ± 0.23 and 4.25 ± 0.48), IL-1β (5.68 ± 1.51 and 12.13 ± 3.71), and IL-18 (2.32 ± 0.37 and 4.81 ± 0.51) compared with controls (all p < 0.005). IMQ stimulation of PBMC from patients similarly induced greater increases in protein expressions of NLRP3 inflammasome compared with controls. The protein expressions of NLRP3, IL-1β, and IL-18 on PBMC significantly decreased after treatment with NLRP3 inhibitor in patients with AOSD.Conclusion.Increased expression of NLRP3 inflammasome and its positive correlation with disease activity in AOSD suggest its involvement in disease pathogenesis. IMQ upregulated expressions of NLRP3 inflammasome signaling, and IMQ might be an activator of NLRP3 inflammasome in AOSD.

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

2020 ◽  
Vol 295 (51) ◽  
pp. 17684-17697 ◽  
Author(s):  
Fang Xu ◽  
Hui Qi ◽  
Jieqiong Li ◽  
Lin Sun ◽  
Juanjuan Gong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nlrp3 Inflammasome ◽  
Mononuclear Cells ◽  
Specific Antigen ◽  
Mycobacterium Tuberculosis Infection ◽  
Peripheral Blood Mononuclear ◽  
Mitofusin 2 ◽  
Pyrin Domain ◽  
Domain 3

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.

scholarly journals Blood functional assay for rapid clinical interpretation of germline TP53 variants

2020 ◽  
pp. jmedgenet-2020-107059 ◽  
Author(s):  
Sabine Raad ◽  
Marion Rolain ◽  
Sophie Coutant ◽  
Céline Derambure ◽  
Raphael Lanos ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Transcriptional Response ◽  
Functional Assay ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Patients With Cancer ◽  
Pathogenic Variants ◽  
Functional Variants ◽  
Multiplex Ligation Probe Amplification ◽  
P53 Mrna

BackgroundThe interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a growing challenge considering the exponential increase in TP53 tests. We developed a functional assay directly performed on patients’ blood.MethodsPeripheral blood mononuclear cells were cultured, activated, exposed to doxorubicin and the p53-mediated transcriptional response was quantified using reverse transcription–multiplex ligation probe amplification and RT-QMPSF assays, including 10 p53 targets selected from transcriptome analysis, and two amplicons to measure p53 mRNA levels. We applied this blood functional assay to 77 patients addressed for TP53 analysis.ResultsIn 51 wild-type TP53 individuals, the mean p53 functionality score was 12.7 (range 7.5–22.8). Among eight individuals harbouring likely pathogenic or pathogenic variants, the scores were reduced (mean 4.8, range 3.1–7.1), and p53 mRNA levels were reduced in patients harbouring truncating variants. We tested 14 rare unclassified variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro191Arg), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del), c.-117G>T) and 12 yielded functionally abnormal scores. Remarkably, the assay revealed that the c.*1175A>C polymorphic variant within TP53 poly-adenylation site can impact p53 function with the same magnitude as a null variant, when present on both alleles, and may act as a modifying factor in pathogenic variant carriers.ConclusionThis blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline TP53 variants and detection of non-coding functional variants.

scholarly journals Decreased levels of miR-28-5p and miR-361-3p and increased levels of insulin-like growth factor 1 mRNA in mononuclear cells from patients with hereditary hemorrhagic telangiectasia

2019 ◽  
Vol 97 (6) ◽  
pp. 562-569 ◽  
Author(s):  
Anthony Cannavicci ◽  
Qiuwang Zhang ◽  
Si-Cheng Dai ◽  
Marie E. Faughnan ◽  
Michael J.B. Kutryk
Keyword(s):  
Growth Factor ◽  
Peripheral Blood ◽  
Hereditary Hemorrhagic Telangiectasia ◽  
Mononuclear Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Manifestations ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Peripheral Blood Mononuclear ◽  
Micro Rnas

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder inherited in an autosomal dominant manner. Patients with HHT can develop vascular dysplasias called telangiectasias and arteriovenous malformations (AVMs). Our objective was to profile and characterize micro-RNAs (miRNAs), short noncoding RNAs that regulate gene expression posttranscriptionally, in HHT patient-derived peripheral blood mononuclear cells (PBMCs). PBMCs, comprised mostly of lymphocytes and monocytes, have been reported to be dysfunctional in HHT. A total of 40 clinically confirmed HHT patients and 22 controls were enrolled in this study. PBMCs were isolated from 16 mL of peripheral blood and purified for total RNA. MiRNA expression profiling was conducted with a human miRNA array analysis. Select dysregulated miRNAs and miRNA targets were validated with reverse transcription–quantitative polymerase chain reaction. Of the 377 miRNAs screened, 41 dysregulated miRNAs were identified. Both miR-28-5p and miR-361-3p, known to target insulin-like growth factor 1 (IGF1), a potent angiogenic growth factor, were found to be significantly downregulated in HHT patients. Consequently, IGF1 mRNA levels were found to be significantly elevated. Our research successfully identified miRNA dysregulation and elevated IGF1 mRNA levels in PBMCs from HHT patients. This novel discovery represents a potential pathogenic mechanism that could be targeted to alleviate clinical manifestations of HHT.

scholarly journals Chronic oral administration of P. gingivalis induces microglial activation and degeneration of dopaminergic neurons possibly through increase in gut permeability and peripheral IL-17A in LRRK2 R1441G mice

2020 ◽  
Author(s):  
Yu-kun Feng ◽  
Yan-Wen Peng ◽  
Qiong-Li Wu ◽  
Feng-Yin Liang ◽  
Hua-Jing You ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Chronic Periodontitis ◽  
Dopaminergic Neurons ◽  
Microglial Activation ◽  
Mononuclear Cells ◽  
Late Onset ◽  
Interleukin 1 ◽  
Peripheral Inflammation ◽  
Peripheral Blood Mononuclear

Abstract Background The R1441G mutation in the leucine-rich repeat kinase 2 (LRRK2) gene results in late-onset Parkinson’s disease (PD). Peripheral inflammation and gut microbiota are closely associated with the pathogenesis of PD. Chronic periodontitis is a common type of peripheral inflammation, which is associated with PD. Porphyromonas gingivalis (Pg), the most common bacterium causing chronic periodontitis, can cause alteration of gut microbiota. It is not known whether Pg-induced dysbiosis plays a role in the pathophysiology of PD. Methods In this study, live Pg were orally administrated to animals, three times a week for one month. Pg-derived lipopolysaccharide (LPS) was used to stimulate peripheral blood mononuclear cells in vitro. The effects of oral Pg administration on the gut and brain were evaluated through behaviors, morphology, and cytokine expression. Results Dopaminergic neurons in the substantia nigra were reduced and activated microglial cells were increased in R1441G mice given oral Pg. In addition, an increase in mRNA expression of tumor necrosis factor (TNF-α) and interleukin-1 β (IL-1β) as well as protein level of α-synuclein together with a decrease in zonula occludens-1 (Zo-1) were detected in the colon in Pg-treated R1441G mice. Furthermore, serum interleukin-17A (IL-17A) and brain IL-17 receptor A (IL-17RA) were increased in Pg-treated R1441G mice. Conclusions These findings suggest that LRRK2 causes gut leakage and further mediates peripheral IL-17A response in Pg-treated animals. We, thus, put forward the hypothesis that IL-17A in the serum may result in activation of the IL-17A-IL-17RA axis that aggravates dysfunction of dopaminergic neurons and provokes microglial activation in LRRK2 R1441G mice.

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