DEPTOR-mTOR Signaling Is Critical for Lipid Metabolism and Inflammation Homeostasis of Lymphocytes in Human PBMC Culture

Abnormal immune response of the body against substances and tissues causes autoimmune diseases, such as polymyositis, dermatomyositis, and rheumatoid arthritis. Irregular lipid metabolism and inflammation may be a significant cause of autoimmune diseases. Although much progress has been made, mechanisms of initiation and proceeding of metabolic and inflammatory regulation in autoimmune disease have not been well-defined. And novel markers for the detection and therapy of autoimmune disease are urgent. mTOR signaling is a central regulator of extracellular metabolic and inflammatory processes, while DEP domain-containing mTOR-interacting protein (DEPTOR) is a natural inhibitor of mTOR. Here, we report that overexpression of DEPTOR reduces mTORC1 activity in lymphocytes of human peripheral blood mononuclear cells (PBMCs). Combination of DEPTOR overexpression and mTORC2/AKT inhibitors effectively inhibits lipogenesis and inflammation in lymphocytes of PBMC culture. Moreover, DEPTOR knockdown activates mTORC1 and increases lipogenesis and inflammations. Our findings provide a deep insight into the relationship between lipid metabolism and inflammations via DEPTOR-mTOR pathway and imply that DEPTOR-mTOR in lymphocytes of PBMC culture has the potential to be as biomarkers for the detection and therapies of autoimmune diseases.

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PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010340 ◽

2019 ◽

Vol 295 (3) ◽

pp. 868-882 ◽

Cited By ~ 1

Author(s):

Nicholas E. Powers ◽

Benjamin Swartzwelter ◽

Carlo Marchetti ◽

Dennis M. de Graaf ◽

Alexandra Lerchner ◽

...

Keyword(s):

Receptor Antagonist ◽

Inflammatory Markers ◽

Mononuclear Cells ◽

Inflammatory Diseases ◽

Human Peripheral Blood ◽

Interleukin 1 ◽

The Body ◽

Random Coil ◽

Peripheral Blood Mononuclear

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS–IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS–IL-1Ra molecules, PAS600–IL-1Ra and PAS800–IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600–IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600–IL-1Ra and PAS800–IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800–IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800–IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600–IL-1Ra, and PAS800-IL-Ra reduced IL-1α–induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans–induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS–IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS–IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

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Transcriptome-Wide Profile of 25-Hydroxyvitamin D3 in Primary Immune Cells from Human Peripheral Blood

Nutrients ◽

10.3390/nu13114100 ◽

2021 ◽

Vol 13 (11) ◽

pp. 4100

Author(s):

Andrea Hanel ◽

Igor Bendik ◽

Carsten Carlberg

Keyword(s):

Vitamin D ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Human Peripheral Blood ◽

Cell Types ◽

Physiological Concentration ◽

Peripheral Blood Mononuclear ◽

Dihydroxyvitamin D3 ◽

Interacting Protein

Vitamin D3 is an essential micronutrient mediating pleiotropic effects in multiple tissues and cell types via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), which activates the transcription factor vitamin D receptor. In this study, we used peripheral blood mononuclear cells (PBMCs) obtained from five healthy adults and investigated transcriptome-wide, whether the precursor of 1,25(OH)2D3, 25-hydroxyvitamin D3 (25(OH)D3), has gene regulatory potential on its own. Applying thresholds of >2 in fold change of gene expression and <0.05 as a false discovery rate, in this ex vivo approach the maximal physiological concentration of 25(OH)D3 (250 nM (nmol/L)) none of the study participants had a significant effect on their PBMC transcriptome. In contrast, 1000 and 10,000 nM 25(OH)D3 regulated 398 and 477 genes, respectively, which is comparable to the 625 genes responding to 10 nM 1,25(OH)2D3. The majority of these genes displayed specificity to the tested individuals, but not to the vitamin D metabolite. Interestingly, the genes MYLIP (myosin regulatory light chain interacting protein) and ABCG1 (ATP binding cassette subfamily G member 1) showed to be specific targets of 10,000 nM 25(OH)D3. In conclusion, 100- and 1000-fold higher 25(OH)D3 concentrations than the reference 10 nM 1,25(OH)2D3 are able to affect the transcriptome of PBMCs with a profile comparable to that of 1,25(OH)2D3.

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Spectrally and Time-Resolved Fluorescence Imaging of 22-NBD-Cholesterol in Human Peripheral Blood Mononuclear Cells in Chronic Kidney Disease Patients

Molecules ◽

10.3390/molecules26226800 ◽

2021 ◽

Vol 26 (22) ◽

pp. 6800

Author(s):

Ingrid Lajdova ◽

Livia Ovsonkova ◽

Viera Spustova ◽

Adrian Oksa ◽

Dusan Chorvat ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Lipid Metabolism ◽

Kidney Disease ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Peripheral Blood Mononuclear ◽

Time Resolved ◽

Blood Mononuclear Cells

The interaction of the fluorescent probe 22-NBD-cholesterol with membranes of human peripheral blood mononuclear cells (PBMC) was tested by time- and spectrally resolved fluorescence imaging to monitor the disturbance of lipid metabolism in chronic kidney disease (CKD) and its treatment with statins. Blood samples from healthy volunteers (HV) and CKD patients, either treated or untreated with statins, were compared. Spectral imaging was done using confocal microscopy at 16 spectral channels in response to 458 nm excitation. Time-resolved imaging was achieved by time-correlated single photon counting (TCSPC) following excitation at 475 nm. The fluorescence of 22-NBD-cholesterol was mostly integrated into plasmatic membrane and/or intracellular membrane but was missing from the nuclear region. The presence of two distinct spectral forms of 22-NBD-cholesterol was uncovered, with significant variations between studied groups. In addition, two fluorescence lifetime components were unmasked, changing in CKD patients treated with statins. The gathered results indicate that 22-NBD-cholesterol may serve as a tool to study changes in the lipid metabolism of patients with CKD to monitor the effect of statin treatment.

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Gut bacteria from multiple sclerosis patients modulate human T cells and exacerbate symptoms in mouse models

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711235114 ◽

2017 ◽

Vol 114 (40) ◽

pp. 10713-10718 ◽

Cited By ~ 275

Author(s):

Egle Cekanaviciute ◽

Bryan B. Yoo ◽

Tessel F. Runia ◽

Justine W. Debelius ◽

Sneha Singh ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Gut Bacteria ◽

The Body ◽

Healthy Controls ◽

Peripheral Blood Mononuclear ◽

Cell Functions ◽

Multiple Sclerosis Patients

The gut microbiota regulates T cell functions throughout the body. We hypothesized that intestinal bacteria impact the pathogenesis of multiple sclerosis (MS), an autoimmune disorder of the CNS and thus analyzed the microbiomes of 71 MS patients not undergoing treatment and 71 healthy controls. Although no major shifts in microbial community structure were found, we identified specific bacterial taxa that were significantly associated with MS. Akkermansia muciniphila and Acinetobacter calcoaceticus, both increased in MS patients, induced proinflammatory responses in human peripheral blood mononuclear cells and in monocolonized mice. In contrast, Parabacteroides distasonis, which was reduced in MS patients, stimulated antiinflammatory IL-10–expressing human CD4+CD25+ T cells and IL-10+FoxP3+ Tregs in mice. Finally, microbiota transplants from MS patients into germ-free mice resulted in more severe symptoms of experimental autoimmune encephalomyelitis and reduced proportions of IL-10+ Tregs compared with mice “humanized” with microbiota from healthy controls. This study identifies specific human gut bacteria that regulate adaptive autoimmune responses, suggesting therapeutic targeting of the microbiota as a treatment for MS.

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Specific TLR4 Blocking Effect of a Novel 3,4-Dihydropyrimidinone Derivative

Frontiers in Pharmacology ◽

10.3389/fphar.2020.624059 ◽

2021 ◽

Vol 11 ◽

Author(s):

Mingqian Zhou ◽

Yiqi Wang ◽

Xiaoying Lin ◽

Jieping Wan ◽

Chengping Wen

Keyword(s):

Molecular Docking ◽

Autoimmune Diseases ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Mrna Levels ◽

Peripheral Blood Mononuclear ◽

Tnf Α ◽

Blood Mononuclear Cells

Background: Toll-like receptor 4 (TLR4) initiates both innate and adaptive immune responses, which plays an important protective role in self-defense mechanisms. Excessive or inappropriate TLR4 activation causes the development of many autoimmune diseases. Dihydropyrimidinone derivatives are medicinally important molecules with diverse pharmacological activities, including anti-inflammatory activity. The present study focused on novel synthesized 3,4-dihydropyrimidinone derivatives and evaluated their inhibitory effects on TLR4.Methods: A series of 3,4-dihydropyrimidinone derivatives were recently synthesized and evaluated for their TLR4 inhibition activities and cytotoxic on HEK-BlueTM hTLR4 cells with the help of QUANTI-Blue assay and MTS assay. Selected compound 3 was analyzed for its molecular docking with TLR4 by using Autodock vina 1.1.2. Its effect on the TLR4 pathway related cytokines was also evaluated in THP-1 cells and human peripheral blood mononuclear cells by using real-time PCR, ELISA and western blot.Results: Five compounds were synthesized and characterized for effectiveness based on 3,4-dihydropyrimidinone. Compound 3 was found to be the potent hybrid among the synthesized compounds, with high TLR4 inhibition activities and low cytotoxic activities against HEK-BlueTM hTLR4 cells. Molecular docking analysis showed that two hydrogen bonds between compound 3 and residues Asp209(TLR4) and Asp99(MD-2) mainly contribute to the TLR4 inhibition. In addition, compound 3 suppressed LPS-induced of the mRNA expression of TLR4, IP-10, TNF-α, IL-6, IL-12A, and IL-12B, the protein expression of pIRF3 and pNFκB and the secretion of IP-10, TNF-α in THP-1 cell line. Compound 3 also inhibited LPS-induced expression of TNF-α, IL-6, and IL-1β but increased IP-10 at mRNA levels in human peripheral blood mononuclear cells.Conclusion: Our study reveals compound 3, a novel 3,4-dihydropyrimidinone derivative, is a potential TLR4 antagonist, which opens up new research avenues for the development of promising therapeutic agents for inflammatory and autoimmune diseases.

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