Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells

Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high-resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Data are available via ProteomeXchange with identifiers PXD001415–23. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced upregulation of proteins such as IL-1β, IL-6, CXCL2, and GROα was confirmed, however, with strong quantitative interindividual differences. Furthermore, the inability of dexamethasone to downregulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. Although most consequences of dexamethasone were found to be compatible with the expected mode of action, some unexpected but significant observations may be related to adverse effects

MOESM4 of Proteomic profiling identifies markers for inflammation-related tumor–fibroblast interaction

Additional file 4. Table S3. GO-term enrichment analysis based on biological process

Proteome Profiling of Breast Cancer Biopsies Reveals a Wound Healing Signature of Cancer-Associated Fibroblasts

Breast cancer is still the most common type of cancer in women; an important role in carcinogenesis is actually attributed to cancer-associated fibroblasts. In this study, we investigated whether it is possible to assess the functional state of cancer-associated fibroblasts through tumor tissue proteome profiling. Tissue proteomics was performed on tumor-central, tumor-near, and tumor-distant biopsy sections from breast adenocarcinoma patients, which allowed us to identify 2074 proteins. Data were interpreted referring to reference proteome profiles generated from primary human mammary fibroblasts comprising 4095 proteins. These cells were analyzed in quiescent cell state as well as after in vitro treatment with TGFβ or IL-1β, stimulating wound healing or inflammatory processes, respectively. Representative for cancer cells, we investigated the mammary carcinoma cell line ZR-75-1, identifying 5212 proteins. All mass analysis data have been made fully accessible via ProteomeXchange, DOI PXD001311 and PXD001323-8. Comparison of tissue proteomics data with all of those reference profiles revealed predominance of cancer cell-derived proteins within the tumor and fibroblast-derived proteins in the tumor-distant tissue sections. Remarkably, proteins characteristic for acute inflammation were hardly identified in the tissue samples. In contrast, several proteins found by us to be induced by TGFβ in mammary fibroblasts, including fibulin-5, SLC2A1, and MUC18, were positively identified in all tissue samples, with relatively higher abundance in tumor neighboring tissue sections. These findings indicate a predominance of cancer-associated fibroblasts with wound healing activities localized around tumors

Extracellular Matrix Remodeling by Bone Marrow Fibroblast-like Cells Correlates with Disease Progression in Multiple Myeloma

The pathogenesis of multiple myeloma (MM) is regarded as a multistep process, in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. Molecular events characteristic for the transition from MGUS to MM are still poorly defined. We hypothesized that fibroblast-like cells in the tumor microenvironment are critically involved in the pathogenesis of MM. Therefore, we performed a comparative proteome profiling study, analyzing primary human fibroblast-like cells isolated from the bone marrow of MM, of MGUS, as well as of non-neoplastic control patients. Thereby, a group of extracellular matrix (ECM) proteins, ECM receptors, and ECM-modulating enzymes turned out to be progressively up-regulated in MGUS and MM. These proteins include laminin α₄, lysyl-hydroxylase 2, prolyl 4-hydroxylase 1, nidogen-2, integrin α₅β₅, c-type mannose receptor 2, PAI-1, basigin, and MMP-2, in addition to PDGF-receptor β and the growth factor periostin, which are likewise involved in ECM activities. Our results indicate that ECM remodeling by fibroblast-like cells may take place already at the level of MGUS and may become even more pronounced in MM. The identified proteins which indicate the stepwise progression from MGUS to MM may offer new tools for therapeutic strategies

Selected identification data obtained from shotgun analysis of cytoplasmic and secretome fractions of HUVECs.

<p>Accession, Uniprot Accession number; Gene, gene name, Peptide IDs, number of distinct peptide identification; C, cytoplasmic fraction; S, secretome fraction. A semi-quantitative abundance measure is provided via emPAI values. Con, untreated cells; IL1b, stimulated with Interleukin 1-beta; m rac, stimulated with Interleukin 1-beta and treated with <i>M rac</i> extract. While inflammatory stimulation increased almost all emPAI values of the listed inflammatory mediators, treatment with <i>M rac</i> extract of stimulated cells resulted in the down-regulation of most emPAI values.</p

2D-PAGE of cytoplasmic proteins isolated from human umbilical vein endothelial cells (HUVECs).

<p>Cells were metabolically labelled while being inflammatory stimulated and treated with <i>M rac</i> extract (1:10’000 dilution), proteins were detected by autoradiography. A) untreated control. B) inflammatory stimulation with interleukin 1-beta strongly induced inflammatory mediators such as MX1, IFIT1, WARS and GBP2 as well as the stress-related protein STIP1. C) treatment of HUVECs with <i>M rac</i> had little effect on the synthesis of cytoplasmic proteins. D) treatment of stimulated HUVECs with <i>M rac</i> extract attenuated the induction of IFIT1, GBP2 and STIP1. Proteins were identified by proteolytic digestion of each spot and subsequent LC-MS/MS analysis.</p

Two-dimensional representation of an LC-MS analysis of the <i>M rac</i> extract.

<p>The analytes were separated using HPLC and detected with a Q Exactive orbitrap in the positive ion mode. Each spot annotates a distinct molecular feature, which can be counted dependent on the applied sensitivity threshold. Several hundred different constituents can easily be distinguished. The x-axis shows the retention time between 10 and 26 min and the y-axis shows the mass range of <i>m/z</i> 150–750.</p

Proteomic and Metabolomic Analyses Reveal Contrasting Anti-Inflammatory Effects of an Extract of Mucor Racemosus Secondary Metabolites Compared to Dexamethasone

<div><p>Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of <i>Mucor racemosus</i> (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424–102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac treatment on lipids were orthogonal to the effect of dexamethasone underlining differences in the overall mode of action.</p></div

Regulation of selected metabolites assessed by the AbsoluteIDQ p180 metabolomics kit illustrating the contrasting inflammation modulating effects of dexamethasone (dex) and M rac.

<p>Again, HUVECs were stimulated with IL-1βeta (IL-1β) and additionally treated with dexamethasone or M rac extract (M rac). Error bars are derived from two technical and two biological replicates. (p-values: *, p<0.05 and **, p<0.005.)</p

Targeted proteomic analysis of pro-inflammatory cytokines secreted by inflammatory stimulated and treated HUVECs.

<p>Experiments were performed using two biological with two technical replicates. Inflammatory stimulation (IL-1β) up-regulated the levels of all cytokines in comparison to untreated controls (con). Treatment with dexamethasone (dex) down-regulated all cytokines as expected for a strong anti-inflammatory drug. Remarkably, also treatment with <i>M rac</i> extract was capable of significantly down-regulating these cytokines (M rac compared to IL-1β: *, p<0.01; **, p<0.001). “Area” refers to the area under the curve of the chromatographic peak in the nLC-MRM experiment of the quantifier transition of each peptide.</p