Proteome Of Monocytes Research Articles

Comprehensive proteomics of monocytes indicates oxidative imbalance functionally related to inflammatory response in chronic kidney disease-related atherosclerosis.

Atherosclerosis-induced cardiovascular events are the leading cause of mortality in chronic kidney disease (CKD) patients. Monocytes are involved in the formation of atherosclerotic plaques and mediate in the overproduction of ROS, promoting inflammation and oxidative stress. However, the relationship between monocytes, inflammation, and oxidative status in CKD-associated atherosclerosis has not been thoroughly investigated. Monocytes and plasma derived from two groups of CKD patients with varying degrees of atherosclerosis and two groups of patients with cardiovascular disease (CVD) and non-CKD atherosclerosis were analyzed. This study was designed to perform a comprehensive proteomic analysis of monocytes in combination with functional bioinformatics. In addition, a targeted investigation of oxidative stress- and inflammatory-related factors to explore CKD-associated atherosclerosis was applied. Dysregulation of proteins involved in lipid oxidation, cell survival, ROS synthesis and metabolism, and inflammatory responses has been revealed. The characteristic disturbances in the monocyte proteome changed with the progression of CKD. A closer examination of oxidative stress's triggers, mediators, and effects on protein and lipid levels showed alterations in the oxidative imbalance between CKD and CVD. CKD monocytes demonstrated a significant increase of oxidized glutathione without changing the level of its reduced form. Evaluation of enzymatic antioxidants, sources of ROS, and modifications caused by ROS also revealed significant alterations between the study groups. In CKD, inflammation and oxidative imbalance correlated and drove each other. However, in CVD, oxidative stress-related factors were associated with each other but not to inflammatory proteins. Moreover, lipid abnormalities were more specific to classical CVD and unrelated to CKD. Such a comprehensive characterization of monocytes and oxidative stress in CKD and CVD patients has never been presented so far. Obtained results support the involvement of distinct mechanisms underlying the acceleration of atherosclerotic and non-atherosclerotic CKD.

Chromatin accessibility differences of human monocyte identify age associated inflammatory heterogeneity.

Abstract Human aging is associated with low grade chronic inflammation termed, inflammaging, which is a significant risk factor for morbidity and mortality in elderly. The innate immune system is master regulator of this process via regulating expression of cytokines and other key mediators. We assayed chromatin landscape of the human monocytes during healthy aging with an aim to predict the inflammation associated contemporary and imminent states of monocytes. Our deep sequencing ATAC-seq data from healthy donors of the GESTALT cohort identified, chromatin landscape signatures defining heterogeneity of monocytes. Differentially open chromatin (DOC) clustered old individual monocytes in two distinct groups, while young donors demonstrated ethnicity-based differences. DOC’s heterogeneity of old donors was characterized by presence of intergenic enhancer-like elements dominated by the DNA binding motif of the transcription factor NF-κB. We identified the relationships of age-associated DOC with DNA methylation, gene expression and proteome of monocytes and serum. Finally, the intracellular cytokine staining analysis using multi-parameter flow cytometry demonstrated the DOC’s influence on the monocyte’s response to LPS stimulation. Our data revels the age associated heterogenous functional states of monocytes which can help us understanding decision making mechanisms during an immune response.

Differential expression of alpha II spectrin in monocytes of tuberculosis patients

Monocytes play a crucial role in immune response to tuberculosis. The present study focuses on identifying differences in the monocyte proteome profile of tuberculosis patients, household contacts and healthy controls. Differential protein expression was studied by two-dimensional (2D) gel electrophoresis. One of the spots consistently showed either lower intensity or was absent in patients and was identified as alpha II-spectrin. The decreased expression of αII-spectrin was further validated by quantitative PCR (qPCR) and western blot analysis. This study suggests the possible role of decreased levels of αII-spectrin in the pathology of tuberculosis.

Proteomic and Systems Biology Analysis of Monocytes Exposed to Securinine, a GABAA Receptor Antagonist and Immune Adjuvant

Securinine, a GABAA receptor antagonist, has been reported to enhance monocyte cell killing of Coxiella burnetii without obvious adverse effects in vivo. We employed multiplex 2D gel electrophoresis using Zdyes, a new generation of covalently linked fluorescent differential protein detection dyes to analyze changes in the monocyte proteome in response to Securinine. Securinine antagonism of GABAA receptors triggers the activation of p38. We used the differential protein expression results to guide a search of the literature and network analysis software to construct a systems biology model of the effect of Securinine on monocytes. The model suggests that various metabolic modulators (fatty acid binding protein 5, inosine 5′-monophosphate dehydrogenase, and thioredoxin) are at least partially reshaping the metabolic landscape within the monocytes. The actin bundling protein L-plastin, and the Ca2+ binding protein S100A4 also appear to have important roles in the immune response stimulated by Securinine. Fatty acid binding protein 5 (FABP5) may be involved in effecting lipid raft composition, inflammation, and hormonal regulation of monocytes, and the model suggests that FABP5 may be a central regulator of metabolism in activated monocytes. The model also suggests that the heat shock proteins have a significant impact on the monocyte immune response. The model provides a framework to guide future investigations into the mechanisms of Securinine action and with elaboration may help guide development of new types of immune adjuvants.

Proteomic profiling of high glucose primed monocytes identifies cyclophilin A as a potential secretory marker of inflammation in type 2 diabetes

Hyperglycemia is widely recognized to be a potent stimulator of monocyte activity, which is a crucial event in the pathogenesis of atherosclerosis. We analyzed the monocyte proteome for potential markers that would enhance the ability to screen for early inflammatory status in Type 2 diabetes mellitus (T2DM), using proteomic technologies. Monocytic cells (THP-1) were primed with high glucose (HG), their protein profiles were analyzed using 2DE and the downregulated differentially expressed spots were identified using MALDI TOF/MS. We selected five proteins that were secretory in function with the help of bioinformatic programs. A predominantly downregulated protein identified as cyclophilin A (sequence coverage 98%) was further validated by immunoblotting experiments. The cellular mRNA levels of cyclophilin A in various HG-primed cells were studied using qRT-PCR assays and it was observed to decrease in a dose-dependent manner. LC-ESI-MS was used to identify this protein in the conditioned media of HG-primed cells and confirmed by Western blotting as well as ELISA. Cyclophilin A was also detected in the plasma of patients with diabetes. We conclude that cyclophilin A is secreted by monocytes in response to HG. Given the paracrine and autocrine actions of cyclophilin A, the secreted immunophilin could be significant for progression of atherosclerosis in type 2 diabetes. Our study also provides evidence that analysis of monocyte secretome is a viable strategy for identifying candidate plasma markers in diabetes.

HIV-1 transforms the monocyte plasma membrane proteome

How HIV-1 affects the monocyte proteome is incompletely understood. We posit that one functional consequence of virus-exposure to the monocyte is the facilitation of protein transformation from the cytosol to the plasma membrane (PM). To test this, cell surface labeling with CyDye fluorophores followed by 2 dimensional differential in-gel electrophoresis (2D DIGE) and liquid chromatography tandem mass spectrometry (LC–MS/MS) was performed. Fifty three percent of HIV-1 induced proteins were PM associated. These were linked, in large measure, to cellular activation and oxidative stress. They included, but not limited to, biliverdin reductase, leukotriene hydrolase A 4, heat shock protein 70, and cystatin B. HIV-1 induced PM protein translocation was associated with cathepsin B- and caspase 9, 3-dependent apoptosis. In contrast, PMA-treated monocytes bypassed caspase 3, 9 pathways and lead to cathepsin B-dependent necrosis. These results demonstrate that HIV-1 uniquely affects monocyte activation and oxidative stress. These do not affect viral infection dynamics but are linked to stress-induced cell death.

Proteome of monocyte priming by lipopolysaccharide, including changes in interleukin-1beta and leukocyte elastase inhibitor

BackgroundMonocytes can be primed in vitro by lipopolysaccharide (LPS) for release of cytokines, for enhanced killing of cancer cells, and for enhanced release of microbicidal oxygen radicals like superoxide and peroxide. We investigated the proteins involved in regulating priming, using 2D gel proteomics.ResultsMonocytes from 4 normal donors were cultured for 16 h in chemically defined medium in Teflon bags ± LPS and ± 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor. LPS-primed monocytes released inflammatory cytokines, and produced increased amounts of superoxide. AEBSF blocked priming for enhanced superoxide, but did not affect cytokine release, showing that AEBSF was not toxic. After staining large-format 2D gels with Sypro ruby, we compared the monocyte proteome under the four conditions for each donor. We found 30 protein spots that differed significantly in response to LPS or AEBSF, and these proteins were identified by ion trap mass spectrometry.ConclusionWe identified 19 separate proteins that changed in response to LPS or AEBSF, including ATP synthase, coagulation factor XIII, ferritin, coronin, HN ribonuclear proteins, integrin alpha IIb, pyruvate kinase, ras suppressor protein, superoxide dismutase, transketolase, tropomyosin, vimentin, and others. Interestingly, in response to LPS, precursor proteins for interleukin-1β appeared; and in response to AEBSF, there was an increase in elastase inhibitor. The increase in elastase inhibitor provides support for our hypothesis that priming requires an endogenous serine protease.

Variation in the monocyte proteome.

We have determined the variability of the monocyte proteome and identified those proteins that demonstrate the greatest variation in the general control population. Monocytes were isolated from 18 healthy (9 male and 9 female) donors ages 18-50 and with no known genetic or blood disorder. A combination of Ficoll-Paque PLUS density centrifugation of cells found in the buffy coat and positive selection with monoclonal antibodies against CD14, coupled to magnetic beads, led to >98% purity of monocytes. A 100,000 g microsomal membrane fraction or 100,000 g supernatant fraction from a control subject was compared to the equivalent fractions from a distinct control subject by two-dimensional differential gel electrophoresis (2D DIGE). Those protein spots that demonstrated Cy3-/Cy5- ratios greater than 2.5-fold in at least one experiment were selected for further statistical analysis. We determined variability for 31 cytosolic and 12 membrane protein spots. Proteins have been identified for 27 of the cytosolic protein spots and 9 of the microsomal membrane protein spots by in-gel digestion with trypsin followed by reverse-phase high-performance liquid chromatography in line with tandem mass spectrometry. We identified 24 distinct monocyte proteins that demonstrated the greatest variability in this general control population. The proteins demonstrating the greatest variance in the cytosolic fraction were enolase-1 and WD (tryptophan-aspartate) repeat-containing protein 1, and in the membrane fraction they were lamin B1 and L-plastin. This study demonstrates the importance of considering variance in the control population when performing future protein profiling comparisons of monocytes derived from disease versus control populations.

Circulating Human Monocytes in the Acute Coronary Syndrome Express a Characteristic Proteomic Profile

We examined the proteome of circulating monocytes of patients with acute coronary syndrome at different times in comparison to that of patients with stable coronary artery disease. On admission, the expression of 18 spot proteins was altered, 10 of which were totally absent. This pattern changed progressively, and at 6 months, there were no differences with the monocyte proteome of stable patients.

High Expression of Antioxidant Proteins in Dendritic Cells

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

Glutamine starvation of monocytes inhibits the ubiquitin–proteasome proteolytic pathway

Peripheral blood monocytes utilize free glutamine (Gln) in addition to glucose as an important energy substrate. Although this demand increases upon activation, monocytes are commonly confronted with decreased plasma Gln during critical illness and thus suffer from Gln-starvation. Here we investigate the influence of Gln-starvation on protein stability and its effects on the monocyte proteome. Gln-starvation caused a reduction of protein degradation which was accompanied by an accumulation of ubiquitin–protein conjugates and a reduction of intracellular ATP. Similar effects were observed under ATP-reducing conditions and in the presence of a proteasome inhibitor. Using two-dimensional gel electrophoresis we identified the IL-1β precursor protein (pIL-1β) as the, by far, most induced protein in endotoxin-treated monocytes. The degradation of the short-lived pIL-1β was strongly reduced during Gln-starvation, while the degradation of the long-lived, constitutively expressed β-actin was less affected. This indicates that although Gln-starvation reduces protein breakdown on the overall proteasome level, it leads to differential changes in the stability of specific proteins. This selective effect is likely to contribute to the immunocompromised state of monocytes commonly observed during critical illness.