Contribution of Human Fibroblasts and Endothelial Cells to the Hallmarks of Inflammation as Determined by Proteome Profiling

Abstract

In order to systematically analyze proteins fulfilling effector functionalities during inflammation, here we present a comprehensive proteome study of inflammatory activated primary human endothelial cells and fibroblasts. Cells were stimulated with interleukin 1-β and fractionated in order to obtain secreted, cytoplasmic and nuclear protein fractions. Proteins were submitted to a data-dependent bottom up analytical platform using a QExactive orbitrap and the MaxQuant software for protein identification and label-free quantification. Results were further combined with similarly generated data previously obtained from the analysis of inflammatory activated peripheral blood mononuclear cells. Applying a false discovery rate of less than 0.01 at both, peptide and protein level, a total of 8370 protein groups assembled from 117,599 peptides was identified; mass spectrometry data have been made fully accessible via ProteomeXchange with identifier PXD003406 to PXD003417.Comparative proteome analysis allowed us to determine common and cell type-specific inflammation signatures comprising novel candidate marker molecules and related expression patterns of transcription factors. Cardinal features of inflammation such as interleukin 1-β processing and the interferon response differed substantially between the investigated cells. Furthermore, cells also exerted similar inflammation-related tasks; however, by making use of different sets of proteins. Hallmarks of inflammation thus emerged, including angiogenesis, extracellular matrix reorganization, adaptive and innate immune responses, oxidative stress response, cell proliferation and differentiation, cell adhesion and migration in addition to monosaccharide metabolic processes, representing both, common and cell type-specific responsibilities of cells during inflammation.

Fig. 1.

Regulation of proteins in HUVEC (A) and NHDF (B) upon inflammatory activation. Differences in LFQ values (logarithmic scale to the base of two) of proteins determined in activated versus control cells, including corresponding p values (logarithmic scale), are represented as volcano plots for each subcellular fraction. Proteins related to angiogenesis and/or ECM organization, as well as proteins related to the IFN response are highlighted as indicated. Proteins that were found to be regulated in the same subcellular fraction of the other cell type, respectively, are designated as well.

Fig. 2.

Quantitative Venn diagrams of proteins regulated in inflammatory activated HUVEC, NHDF, and PBMCs demonstrating the cell type-specificity of the inflammatory response. For each kind of cell, the number of protein groups found to be at least 2-fold up- or downregulated (p ≤ 0.05) upon inflammatory activation of the cells is represented. In total, we identified 667 protein groups that were regulated in HUVEC, 894 in NHDF and 646 in PBMCs.

Fig. 3.

Cell type-specific regulation of IL-1β, IL-1RA, TGFβ2, and of proteins related to the IFN response. A, Heat maps of LFQ values for IL-1β, IL-1RA, and TGFβ2 are represented, as well as differences of LFQ values for these proteins between control and activated cells (Δ act versus con; logarithmic scale to the base of two) with corresponding p values (logarithmic scale) determined in the cytoplasm (cyt) and the supernatant (sn) of the cells. Acc.Nr., UniProt accession number. B, Heat maps of LFQ values for different IFN-responsive gene products are represented.

Fig. 4.

Cell type-specific regulation of proteins during inflammation. Heat maps of LFQ values for the canonical transcription factors NFκB and AP1, as well as for corresponding target gene products that were up-regulated in a cell type-specific way in HUVEC, NHDF, and PBMCs, are represented. LFQ values for cell type-specific transcription factors with target gene products are shown similarly.

Fig. 5.

Inflammation-related processes realized by all cells, however, by upregulating different proteins. Heat maps of LFQ values for proteins involved in the innate immune response, cell adhesion and migration, cell proliferation and differentiation as well as response to oxidative stress are represented.

Fig. 6.

Hallmarks of inflammation. Hallmarks of inflammation representing the most apparent biological processes executed by HUVEC, NHDF, and/orPBMCs are represented.