Quantitative proteomics analyses of activation states of human THP-1 macrophages.

Macrophages display large functional and phenotypical plasticity. They can adopt a broad range of activation states depending on their microenvironment. Various surface markers are used to characterize these differentially polarized macrophages. However, this is not informative for the functions of the macrophage. In order to have a better understanding of the functional changes of macrophages upon differential polarization, we studied differences in LPS- and IL4-stimulated macrophages. The THP-1 human monocytic cell line, was used as a model system. Cells were labeled, differentiated and stimulated with either LPS or IL-4 in a quantitative SILAC proteomics set-up. The resulting sets of proteins were functionally clustered. LPS-stimulated macrophages show increased secretion of proinflammatory peptides, leading to increased pressure on protein biosynthesis and processing. IL4-stimulated macrophages show upregulation of cell adhesion and extracellular matrix remodeling. Our approach provides an integrated view of polarization-induced functional changes and proves useful for studying functional differences between subsets of macrophages. Moreover, the identified polarization specific proteins may contribute to a better characterization of different activation states in situ and their role in various inflammatory processes.

Cell-based screening assay for anti-inflammatory activity of bioactive compounds.

Excess dietary intake may induce metabolic inflammation which is associated with insulin resistance and cardiovascular disease. Recent evidence indicates that dietary bioactive compounds may diminish metabolic inflammation. To identify anti-inflammatory bioactives, we developed a screening assay using the human H293-NF-κB-RE-luc2P reporter cell line. Under optimised conditions we determined the anti-inflammatory activity of vegetables and purified bioactives, by monitoring their potency to inhibit TNF-α-induced NF-κB activity, as assessed by sensitive chemiluminescence detection in a 96-well assay format. Minced broccoli seedlings reduced NF-κB activity by 16%, while sulphoraphane, the dominant bioactive in broccoli seedlings, inhibited NF-κB activity with an IC50 of 5.11 µmol/l. Short-chain fatty acids also reduced NF-κB activity in the order butyrate>propionate≫acetate with IC50 of 51, 223, and 1300 µmol/l, respectively. The H293-NF-κB-RE-luc2P reporter cell line is a sensitive tool for rapid high-throughput screening for bioactives with anti-inflammatory activity.

Clinical outcome, proteome kinetics and angiogenic factors in serum after thermoablation of colorectal liver metastases.

BACKGROUND: Thermoablation is used to treat patients with unresectable colorectal liver metastases (CRLM). We analyze clinical outcome, proteome kinetics and angiogenic markers in patients treated by cryosurgical ablation (CSA) or radiofrequency ablation (RFA). METHODS: 205 patients underwent CSA (n = 20), RFA (n = 22), partial hepatectomy (PH, n = 134) or were found truly unresectable (n = 29). Clinical outcome, proteome transitions and angiogenic response in serum were analyzed at various time points after ablation. RESULT: Median overall survival in CSA patients (17.6 months) was worse (p < 0.0001) when compared to RFA treated patients (51.7 months) and patients after PH (43.4 months). The complication rate was higher in the CSA group (50%) as compared to the RFA group (22%). Proteomics analyses showed consistently more changes in serum protein abundance with CSA compared to RFA. In the first four days after ablation a pro-angiogenic serum response occurred. CONCLUSIONS: RFA of CRLM is superior to CSA with a median survival which equals survival in patients after PH. Proteomics analyses suggests a more aggravated serum response to CSA compared to RFA. Thermoablation is associated with changes in serum levels of angiogenic factors favouring a pro-angiogenic environment, but without differences between RFA and CSA.

Short-chain fatty acids stimulate angiopoietin-like 4 synthesis in human colon adenocarcinoma cells by activating peroxisome proliferator-activated receptor γ.

Angiopoietin-like protein 4 (ANGPTL4/FIAF) has been proposed as a circulating mediator between the gut microbiota and fat storage. Here, we show that transcription and secretion of ANGPTL4 in human T84 and HT29 colon adenocarcinoma cells is highly induced by physiological concentrations of short-chain fatty acids (SCFA). SCFA induce ANGPTL4 by activating the nuclear receptor peroxisome proliferator activated receptor γ (PPARγ), as demonstrated using PPARγ antagonist, PPARγ knockdown, and transactivation assays, which show activation of PPARγ but not PPARα and PPARδ by SCFA. At concentrations required for PPARγ activation and ANGPTL4 induction in colon adenocarcinoma cells, SCFA do not stimulate PPARγ in mouse 3T3-L1 and human SGBS adipocytes, suggesting that SCFA act as selective PPARγ modulators (SPPARM), which is supported by coactivator peptide recruitment assay and structural modeling. Consistent with the notion that fermentation leads to PPAR activation in vivo, feeding mice a diet rich in inulin induced PPAR target genes and pathways in the colon. We conclude that (i) SCFA potently stimulate ANGPTL4 synthesis in human colon adenocarcinoma cells and (ii) SCFA transactivate and bind to PPARγ. Our data point to activation of PPARs as a novel mechanism of gene regulation by SCFA in the colon, in addition to other mechanisms of action of SCFA.

Propionic acid affects immune status and metabolism in adipose tissue from overweight subjects.

BACKGROUND: Adipose tissue is a primary site of obesity-induced inflammation, which is emerging as an important contributor to obesity-related diseases such as type 2 diabetes. Dietary fibre consumption appears to be protective. Short-chain fatty acids, e.g. propionic acid, are the principal products of the colonic fermentation of dietary fibre and may have beneficial effects on adipose tissue inflammation. MATERIALS AND METHODS: Human omental adipose tissue explants were obtained from overweight (mean BMI 28·8) gynaecological patients who underwent surgery. Explants were incubated for 24 h with propionic acid. Human THP-1 monocytic cells were differentiated to macrophages and incubated with LPS in the presence and absence of propionic acid. Cytokine and chemokine production were determined by multiplex-ELISA, and mRNA expression of metabolic and macrophages genes was determined by RT-PCR. RESULTS: Treatment of adipose tissue explants with propionic acid results in a significant down-regulation of several inflammatory cytokines and chemokines such as TNF-α and CCL5. In addition, expression of lipoprotein lipase and GLUT4, associated with lipogenesis and glucose uptake, respectively, increased. Similar effects on cytokine and chemokine production by macrophages were observed. CONCLUSION: We show that propionic acid, normally produced in the colon, may have a direct beneficial effect on visceral adipose tissue, reducing obesity-associated inflammation and increasing lipogenesis and glucose uptake. Effects on adipose tissue as a whole are at least partially explained by effects on macrophages but likely also adipocytes are involved. This suggests that, in vivo, propionic acid and dietary fibres may have potential in preventing obesity-related inflammation and associated diseases.

Regulation of adipokine production in human adipose tissue by propionic acid.

BACKGROUND: Dietary fibre (DF) has been shown to be protective for the development of obesity, insulin resistance and type 2 diabetes. Short-chain fatty acids, produced by colonic fermentation of DF might mediate this beneficial effect. Adipose tissue plays a key role in the regulation of energy homeostasis, therefore, we investigated the influence of the short-chain fatty acid propionic acid (PA) on leptin, adiponectin and resistin production by human omental (OAT) and subcutaneous adipose tissue (SAT). As PA has been shown to be a ligand for G-protein coupled receptor (GPCR) 41 and 43, we investigated the role of GPCR's in PA signalling. MATERIALS AND METHODS: Human OAT and SAT explants were obtained from gynaecological patients who underwent surgery. Explants were incubated for 24 h with PA. Adipokine secretion and mRNA expression were determined using ELISA and RT-PCR respectively. RESULTS: We found that PA significantly stimulated leptin mRNA expression and secretion by OAT and SAT, whereas it had no effect on adiponectin. Furthermore, PA reduced resistin mRNA expression. Leptin induction, but not resistin reduction, was abolished by inhibition of Gi/o-coupled GPCR signalling. Moreover, GPCR41 and GPCR43 mRNA levels were considerably higher in SAT than in OAT. CONCLUSIONS: We demonstrate that PA stimulates expression of the anorexigenic hormone leptin and reduces the pro-inflammatory factor resistin in human adipose tissue depots. This suggests that PA is involved in regulation of human energy metabolism and inflammation and in this way may influence the development of obesity and type 2 diabetes.

Computational modeling of the human serum proteome response to colon resection surgery.

Principal component analysis (PCA) is much used in exploring time-course biological data sets, but does not distinguish variation between time and subjects. This study proposes a new integrated approach by combining analysis of variance (ANOVA) and three component modeling methods. The former was used to separate the between- and within-subject variation, and the latter represent modeling strategies on a scale moving from commonality to individuality. The proposed approach was applied to a surface-enhanced laser desorption and ionization time of flight mass spectrometry (SELDI-TOF-MS) data set of a serum protein expression time course before and after colon resection. Two common biological processes are identified and individual differences among patients were also detected, and the biological relevance of both is discussed.

Proteomics analysis of Hodgkin lymphoma: identification of new players involved in the cross-talk between HRS cells and infiltrating lymphocytes.

Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) secrete factors that interact with inflammatory background cells and may serve as biomarkers for disease activity. To detect new proteins related to pathogenesis, we analyzed the secretome of HRS cells. Proteins in cell culture supernatant of 4 HL cell lines were identified using 1DGE followed by in-gel trypsin digestion and LC-MS/MS. In total, 1290 proteins, including 368 secreted proteins, were identified. Functional grouping of secreted proteins revealed 37 proteins involved in immune response. Sixteen of the 37 proteins (ie, ALCAM, Cathepsin C, Cathepsin S, CD100, CD150, CD26, CD44, CD63, CD71, Fractal-kine, IL1R2, IL25, IP-10, MIF, RANTES, and TARC) were validated in HL cell lines and patient material using immunohistochemistry and/or ELISA. Expression of all 16 proteins was confirmed in HL cell lines, and 15 were also confirmed in HL tissues. Seven proteins (ALCAM, cathepsin S, CD26, CD44, IL1R2, MIF, and TARC) revealed significantly elevated levels in patient plasma compared with healthy controls. Proteomics analyses of HL cell line supernatant allowed detection of new secreted proteins, which may add to our insights in the interaction between HRS cells and infiltrating lymphocytes and in some instances might serve as biomarkers.

Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series.

Monitoring changes in serum protein expression in response to acute events such as trauma, infection or drug intervention may reveal key proteins of great value in predicting recovery or treatment response. Concerted actions of many proteins are expected. Proteins sharing similar expression changes may function in the same physiological process. As a model we analyzed expression changes in serum of colon cancer patients, before, during, and after laparoscopic colon resection. Eight samples were taken from each of four patients before, during, and up to 5 days after surgery. Total serum and a low molecular weight fraction were analyzed by SELDI-TOF-MS. In total 146 masses were detected. A principal components analysis (PCA) illustrates the temporal variation in the postsurgery proteome. Time series for each mass could be clustered into four distinct groups based on similarity in expression pattern. Two masses of 11.4 and 11.6 kDa, part of a slow response cluster, were identified as forms of the acute phase protein serum amyloid A (SAA). Fourteen more proteins belong to this cluster and may also function in acute phase response. We present an approach to analyze temporal variation in the proteome. This approach may be useful to evaluate surgical, nutritional, and pharmacological interventions.

Proteomic analyzes of copper metabolism in an in vitro model of Wilson disease using surface enhanced laser desorption/ionization-time of flight-mass spectrometry.

In Wilson disease, mutations in the ATP7B-gene lead to hepatic accumulation of copper that becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and acute liver failure. Several proteins are probably involved in dealing with the excess copper and oxidative stress. As a first step towards biomarker discovery and analyzes of copper metabolism in Wilson disease patients we characterized copper-induced changes in protein expression in cell lysates and culture media from an in vitro copper-overload model using surface enhanced laser desorption/ionization (SELDI) proteomics technology. HepG2 cells were cultured for 48 h with a physiological (0.5 microM) or a pathological (100 microM) copper concentration. Samples were applied to weak cation exchange (WCX) proteinchip arrays and chips were analyzed by time of flight (TOF)-mass spectrometry. Copper-coated IMAC chips were used to detect copper-binding proteins in cell lysate of copper depleted cells using buffers with increasing imidazole concentrations. Data from the 2 to 50 kDa range indicate that high extra-cellular copper substantially altered both intra-cellular protein expression as well as the composition of the secretome. In the lysate 15 proteins were found up-regulated, while 6 proteins were down-regulated. In culture media 21 proteins were increased while 4 proteins were decreased in abundance. Copper-coated protein chips revealed the presence of 18 high-affinity copper-binding proteins. Further identification is necessary to determine the exact cellular roles of the discovered proteins.