Pathways In Mononuclear Cells Research Articles

Regulation of S100As Expression by Inflammatory Cytokines in Chronic Lymphocytic Leukemia.

The calcium-binding proteins S100A4, S100A8, and S100A9 are upregulated in chronic lymphocytic leukemia (CLL), while the S100A9 promotes NF-κB activity during disease progression. The S100-protein family has been involved in several malignancies as mediators of inflammation and proliferation. The hypothesis of our study is that S100A proteins are mediators in signaling pathways associated with inflammation-induced proliferation, such as NF-κB, PI3K/AKT, and JAK/STAT. The mononuclear cells (MNCs) of CLL were treated with proinflammatory IL-6, anti-inflammatory IL-10 cytokines, inhibitors of JAK1/2, NF-κB, and PI3K signaling pathways, to evaluate S100A4, S100A8, S100A9, and S100A12 expression as well as NF-κB activation by qRT-PCR, immunocytochemistry, and immunoblotting. The quantity of S100A4, S100A8, and S100A9 positive cells (p < 0.05) and their protein expression (p < 0.01) were significantly decreased in MNCs of CLL patients compared to healthy controls. The S100A levels were generally increased in CD19+ cells compared to MNCs of CLL. The S100A4 gene expression was significantly stimulated (p < 0.05) by the inhibition of the PI3K/AKT signaling pathway in MNCs. IL-6 stimulated S100A4 and S100A8 protein expression, prevented by the NF-κB and JAK1/2 inhibitors. In contrast, IL-10 reduced S100A8, S100A9, and S100A12 protein expressions in MNCs of CLL. Moreover, IL-10 inhibited activation of NF-κB signaling (4-fold, p < 0.05). In conclusion, inflammation stimulated the S100A protein expression mediated via the proliferation-related signaling and balanced by the cytokines in CLL.

300 PCSK9 induces a prothrombotic response by activation of NFKB signalling pathway in mononuclear cells

Abstract Aims Strong evidence both experimental and clinical studies point out to an involvement of proprotein convertase subtilisin/kexin 9 (PCSK9), as an important player in hypercholesterolemia and atherosclerosis pathophysiology, identifying it, as a key molecule in the development of new cholesterol-lowering drugs and therapeutic target for atherosclerosis and related diseases. Emerging evidence shown that PCSK9 is straight implicated in inflammatory process where it may directly influence the activity of various cell types through NFkB activation, a key transcription factor involved in the induction of both pro-inflammatory cytokines and Tissue Factor (TF) expression, a major regulator of haemostasis and thrombosis in monocytes. Deductive reasoning makes therefore plausible to investigate a mechanistic link between circulation PCSK9 levels and TF expression, both in monocytes as well as on the surface of microparticles (MPs) generated from the same cells, with NFkB acting as the chain in the link. To investigate the involvement of NFkB signalling pathway in PCSK9-induced TF expression. Methods THP-1 cell line were stimulated with human (h) PCSK9 (5 μg/mL) or pre-incubated with BAY-117082 (BAY, 10−5M) an NFκB inhibitor, CLI-095 (3 × 10−6M), a highly TLR-4 signalling specific inhibitor and LPS-RS(5 μg/mL) a TLR-4 antagonist. TF procoagulant activity (PCA) was assessed by one-stage clotting assay using a STart Max semi-automated coagulation analyser. Concentration of TF-bearing MPs was determined using the Zymuphen MP-activity kit. Results hPCSK9 stimulated TF activity in THP-1 (PCA: from 50 ± 20 to 120 ± 20 ρg/mL, n = 10, P &amp;lt; 0.01). BAY, an NFkB inhibitor (PCA: −71 ± 23%, n = 5, P &amp;lt; 0.01) and CLI-095, a TLR-4 signalling inhibitor, (PCA: −86 ± 26%, n = 3, P &amp;lt; 0.05) and LPS-RS (PCA: −71 ± 23%, n = 5, P &amp;lt; 0.01) down-regulated PCSK9-induced TF activity completely. Furthermore THP-1stimulation with hPCSK9 causes the release of prothrombotic MPs-TF+ (MPs release from: 0.13 ± 0.07 to 0.42 ± 0.1 nM PS, n = 7, P &amp;lt; 0.05; PCA-MP+ from: 14 ± 3 to 44 ± 28 ρg/mL, n = 5, P &amp;lt; 0.05). Conclusions These data confirm the pivotal role of monocytes in the response PCSK9-induced TF-expression as well as the involvement of PCSK9 in inflammatory-thrombotic diseases through a direct stimulation of TF procoagulant activity and by the release of TF-bearing MPs. The possible mechanism of action involves recognition of PCSK9 by TLRs 2 and/or 4, on monocytes membrane surface, leading to activation of the transcription factor NFκB via an intracellular signalling cascade. Further studies will be needed to better understand the regulatory mechanisms underlying this complex set of biological responses that bind PCSK9, and coagulation events.

Exercise training modulates the gut microbiota profile and impairs inflammatory signaling pathways in obese children

Childhood obesity has reached epidemic levels and is a serious health concern associated with metabolic syndrome, nonalcoholic fatty liver disease, and gut microbiota alterations. Physical exercise is known to counteract obesity progression and modulate the gut microbiota composition. This study aims to determine the effect of a 12-week strength and endurance combined training program on gut microbiota and inflammation in obese pediatric patients. Thirty-nine obese children were assigned randomly to the control or training group. Anthropometric and biochemical parameters, muscular strength, and inflammatory signaling pathways in mononuclear cells were evaluated. Bacterial composition and functionality were determined by massive sequencing and metabolomic analysis. Exercise reduced plasma glucose levels and increased dynamic strength in the upper and lower extremities compared with the obese control group. Metagenomic analysis revealed a bacterial composition associated with obesity, showing changes at the phylum, class, and genus levels. Exercise counteracted this profile, significantly reducing the Proteobacteria phylum and Gammaproteobacteria class. Moreover, physical activity tended to increase some genera, such as Blautia, Dialister, and Roseburia, leading to a microbiota profile similar to that of healthy children. Metabolomic analysis revealed changes in short-chain fatty acids, branched-chain amino acids, and several sugars in response to exercise, in correlation with a specific microbiota profile. Finally, the training protocol significantly inhibited the activation of the obesity-associated NLRP3 signaling pathway. Our data suggest the existence of an obesity-related deleterious microbiota profile that is positively modified by physical activity intervention. Exercise training could be considered an efficient nonpharmacological therapy, reducing inflammatory signaling pathways induced by obesity in children via microbiota modulation.

CONDITION OF RIG-IAND NF-KB-SIGNAL PATHWAYS IN MONONUCLEAR CELLS OF WHOLE BLOOD OF PRACTICALLY HEALTHY PEOPLE AND RECONVENCENTS OF PNEUMONIA AFFECTED BY MITOGENIC STIMULATION

The key role in the antiviral and antimicrobial defense of the body is played by the RIG-I and NF-kB signaling pathways. The RIG-I signaling pathway activates interferon-regulated factors IRF3 and IRF7, and the central component of the NF-kB signaling pathway, the nuclear transcription factor NF-kB, determines the production of endogenous antimicrobial peptides and interferons by cells. One of the key regulators of the RIG-I signaling pathway is the mitochondrial protein MAVS, which integrates signals from receptors that recognize pathogenicity patterns. The influence of various factors, such as bacterial toxins, free radicals, reactive oxygen species, leads to MAVS dysfunction, impaired antiviral resistance and the progression of viral infection. However, despite the important role of the proteins of the RIG-I-pathway and the components of the NF-KB-signaling pathway in ensuring the body's resistance to infections and sanogenesis, their significance in the postclinical phase has not been fully studied. The aim of the study was to evaluate the content of the components of RIG-I and NF-kB signaling pathways in mononuclear cells of whole blood of healthy individuals and pneumonia convalescents after exposure to a complex mitogen. The enzyme-linked immunosorbent assay determined the content of components of the NF-kB signaling pathway (p50, p65, c-Rel, RelB, NF-kB2), protein kinases of the NF-kB nuclear transcription factor inhibitor (IkB), and RIG-I- proteins in mononuclear cells of whole blood the signal path (TAK1, TBK1, TRIM25, TMEM173, RNF125, IRF3, IRF7, MAVS), RIG-I-dependent helicase (LGP2), the level of phosphorylation of protein kinase p38 and IkB, as well as the production of whole blood cells IL-4, IL-, were evaluated 12, RANTES, cathelicidin and interferons (IFN-p and IFNa). It was established that in the subclinical phase of community-acquired bacterial pneumonia in mononuclear cells of whole blood after stimulation with a complex mitogen containing lipopolysaccharide, the content of RelB, MaVS, DHX58, and IRF7 decreased compared to practically healthy individuals, p38 protein kinase dephosphorylation was noted. In contrast, the concentrations of IKKa, IKKp, the level of phosphorylation of kBa, the protein content of TRIM25, TMEM173, OTuD5, RNF125 and tBk1 were increased. These changes were accompanied by a statistically significant decrease in the production of IL-4, IL-12, RANTES, cathelicidin and IFN-p against the background of an increase in the level of IFNa. The effect on the mononuclear cells of whole blood of a complex mitogen led to a change in the ratio of the components of the signaling pathways that determine the antibacterial and antiviral defense of the body. In patients with pneumonia, against the background of mitogenic stimulation, the production of cathelicidin,

3448 Macrophages, APOL1 Genotype, &amp; Immunometabolism in CVD (MAGIC

OBJECTIVES/SPECIFIC AIMS: This study aims to understand the potential immunomudualtory effect of APOL1 variants in auto-antigen activated myeloid cells by assessing lysosomal integrity in activated cells expressing APOL1. The primary stimuli were: 1. ssRNA hY3 as a proxy for the Ro immune complex; 2. in an bulk RNA seq model, interferon-response gene, Siglec 1, as a read out of interferon activity. The primary outcomes were: 1. Myeloid cell APOL1 expression both in primary macrophage cultures and ex-vivo patient derived macrophages; 2. Lysosome integrity as measured by fluorescence intensity of lysotracker dye on light microscopy. METHODS/STUDY POPULATION: All recruited subjects provided written informed consent as per the NEW YORK UNIVERSITY Division of Rheumatology-wide Specimen and Matched Phenotype Linked Examination (SAMPLE) protocol. Subjects were African American; SLE subjects met 4 American College of Rheumatology criteria for SLE. Healthy donor monocytes representing each genotype in duplicate (reference allele: G0/G0; heterozygote variant: RV/G0; and homozygote variant RV/RV) were cultured with GM-CSF to yield macrophages which were incubated in serum free media or with hY3 ssRNA (TLR 7/8 agonist) to yield inflammatory M1 macrophages. Fold increase of APOL1 in untreated vs hY3 treated macrophages was measured using qPCR. Live cells were then cultured on glass chamber slides with DNA dye, DAPI, and Lysotracker red, a fluorescent dye that stains acidic lysosomes. As a proof of concept, interferon response gene, Signlec1, and APOL1 transcriptional activity in peripheral blood monocytes (PBMCs) were measured and correlated in 17 SLE patients by RNA seq. RESULTS/ANTICIPATED RESULTS: Regardless of genotype, hY3 increased APOL1 expression by 29 (+/−18.4) fold (P = 0.007 vs no treatment). Genotyping of the qPCR product showed concordance with the chromosomal DNA with the RV heterozygotes expressing both alleles. To examine lysosomal membrane integrity, live hY3-treated macrophages were stained with lysosotracker dye and fluorescence intensity was measured. Compared to reference allele carrying macrophages, each additional variant allele corresponded with a lesser degree of lysosome compartment staining. In SLE PBMCs, we found that APOL1 was highly expressed, and significantly correlated with Siglec1 (F=10.5; P = 0.005) supporting an association between circulating interferons and APOL1 accumulation in monocytes. DISCUSSION/SIGNIFICANCE OF IMPACT: Given that the “cytokine milieu” in SLE elicits APOL1 expression, induces inflammatory cell metabolic rewiring, and stimulates autophagy thereby exposing defects in autophagic flux, this gene-environment interaction may underpin the relationship between chronic inflammation and heightened APOL1 polymorphism-attributed cardiovascular risk. These data support further inquiry into the intersection between chronic autoimmunity and APOL1’s functional role in the vascular microenvironment. The in vitro studies herein extend our prior work by demonstrating a mechanistic link between SLE-associated inflammation, APOL1 risk variant status and CVD via a lysosomal defect which converges on common autophagic and metabolic pathways in mononuclear cells.

Dysregulation of micro-RNAs involved in TLR pathway in peripheral blood mononuclear cells and synovial fluid mononuclear cells of patients with enthesitis related arthritis form of juvenile idiopathic arthritis (HUM3P.254)

Abstract Toll like receptor (TLR) pathway plays a major role in inflammatory arthritis. MicroRNA (miR)-146a, miR-155, miR-21 regulate downstream molecules of TLR signaling like IRAK-1 and TRAF-6. TLR pathway genes are generally upregulated in ERA-JIA. We studied expression of miR-146a, miR-155, miR-21, miR-210 and miR-26a in PBMC and SFMC of patients with ERA. RNA was isolated from PBMC (n=21) &amp; SFMC (n=11) of ERA patients and PBMC of controls (n=11). The miRNA and target gene expressions were analysed by TaqMan qRT-PCR assays. Among 21 patients, 20 were boys. Mean disease onset age was 10.3Y &amp; disease duration was 6Y. All had arthritis, 14 had enthesitis, 4 had uveitis, 7 had IBP and 2 had family history of HLAB27 related disease. No expression difference was seen in miR-146a, miR-155, miR-210 and miR-26a in PBMC of patients and controls but miR-21 was increased in patients. To check inflammatory site specific expression, comparison of SFMC and PBMC was done showing downregulation of miR146a and upregulation of miR-155, miR-21 and miR-210 in SFMC. Expression of miR-26a did not show any change. MiR-146a target genes i.e. IRAK-1 and TRAF-6 were upregulated in SFMC compared to PBMC. There is dysregulation of miRNAs regulating TLR pathway in mononuclear cells of patients with ERA-JIA especially at the chronic inflammation site. Down regulation of miR-146a, a key regulator may lead to over-expression of TLR downstream molecules like IRAK-1 and TRAF-6 thus perpetuating inflammation in ERA.

Investigation of the caspase-dependent mitochondrial apoptotic pathway in mononuclear cells of patients with systemic Lupus erythematosus.

BackgroundThis study aimed to explore the role of apoptosis initiators, caspase-9, caspase-10, mitochondrial anti-viral signaling protein (MAVS), and interferon regulatory factor 7 (pIRF7), in patients with systemic lupus erythematosus (SLE).MethodsLeukocyte apoptosis was determined by flow cytometry, including annexin V, APO2.7, and 7-amino-actinomycin D (7-AAD) on each subtype of leukocyte in 35 patients with SLE, 15 disease controls, and 17 volunteer normal controls. Levels of caspase-9, caspase-10, MAVS, and pIRF7 in mononuclear cells and the disease activity index (SLEDAI) in the SLE patients were determined. Correlation among intracellular adaptor proteins and caspase levels were calculated.ResultsThe SLE patients had higher APO2.7 in total leukocyte, lymphocyte, and monocytes, and higher late apoptosis markers in total leukocytes and neutrophils than normal controls (all p < 0.05). Disease activity was positively associated with the APO2.7 of CD19+ cells in SLE, but negatively associated with MAVS and caspase-9 levels (all p < 0.05). Markers of viral infection and anti-virus transcription factors like MDA5, MAVS, and pIRF7 were significantly higher in SLE patients than in disease controls (p < 0.05). Caspase-9 and caspase-10 levels positively correlated with MAVS and pIRF7 in SLE patients (p < 0.05).ConclusionsThe disease activity of SLE is positively associated with APO2.7 level of CD19+ cells but negatively associated with MAVS and caspase-9 levels, which all point to a mitochondrial pathway.

Activation of the Purine Salvage Pathway in Mononuclear Cells of Cardiac Recipients Treated with Mycophenolate Mofetil

The objective of this study was to investigate purine nucleotide metabolism in peripheral blood mononuclear cells (PBMC) of cardiac transplant recipients switched from azathioprine to mycophenolate mofetil (MMF). Concentrations of guanosine 5'triphosphate (GTP) and adenosine 5'triphosphate (ATP), the activities of inosine monophosphate dehydrogenase (IMPDH), guanine phosphoribosyltransferase (GPRT), and hypoxanthine phosphoribosyltransferase (HPRT) were determined in PBMC of 27 cardiac transplant recipients before switch to MMF and 3, 6, and 12 months thereafter. There was no difference in the activities of IMPDH and salvage pathway enzymes GPRT and HRPT as well as in intracellular GTP and ATP concentrations between the patients before switch to MMF and healthy controls. The GTP and ATP concentrations in PBMC of cardiac recipients did not change during the entire observation period. Although the MPA trough level remained similar, IMPDH activity declined from 897 to 316 pmol/10(6)PBMC/h 3 months after MMF onset, was almost completely inhibited after 6 months, and partially restored to 143 pmol/10(6)PBMC/h 12 months after switch to MMF. In contrast, GPRT activity increased after 3, 6, and 12 months of MMF therapy and HPRT activity 3 and 6 months after switch to MMF. We demonstrated for the first time an induction of salvage pathway enzyme activities in PBMC under MMF therapy. This probably accounts for the maintenance of intracellular purine nucleotide pools and prevents the GTP depletion.

Oxidized Low Density Lipoprotein Increases U937 Cell 5-Lipoxygenase Activity: Induction of 5-Lipoxygenase Activating Protein

This study examines the effects of oxidized low density lipoprotein (ox-LDL) on mononuclear cell arachidonic acid (AA) metabolism. U937 and HL60 cells, employed as models for mononuclear cells and promyelocytic cells, respectively, were exposed to ox-LDL (20 μg protein/ml) for 24 hours. HPLC analysis of 1- 14C-AA metabolites indicates that ox-LDL increases U937 and HL60 cell production of 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). Northern analysis indicates that ox-LDL increases U937 cell FLAP transcript levels 10-times control levels but did not appear to alter 5-lipoxygenase (5-LO) mRNA levels. In contrast, ox-LDL increases HL60 cell transcript levels for FLAP and 5-LO 1.5 times and 10 times control levels, respectively. Thus, we propose that ox-LDL plays an important role in the up-regulation of the 5-LO pathway in mononuclear cells. Such activation may explain, in part, the mechanisms by which ox-LDL promotes atherogenesis.