Proteome bioprofiles distinguish between M1 priming and activation states in human macrophages

Abstract

Macrophage activation is a dynamic process that results in diverse functional outcomes ranging from immunoregulation to inflammation. The proinflammatory, or M1, response is a complex, bimodal progression composed of a "prime," classically through IFN-gamma, and "trigger," such as LPS. To characterize the physiological response of M1 activation, a systems biology approach was applied to determine the intracellular proteome bioprofiles of IFN-gamma-and LPS-treated primary human macrophages. Our goal was to develop intracellular proteomic fingerprints to serve as novel correlates of macrophage priming and/or activation to augment the existing approaches of analyzing secreted cytokines and cell-surface protein expression. The majority of the proteome, approximately 78%, remained stable during activation, representing the core proteome. In contrast, three distinct patterns defined response proteomes: IFN-gamma-specific, LPS-specific, or IFN-gamma- and LPS-shared or M1-specific. Although steady-state expression levels of proteins involved in energy metabolism and immune response were increased during priming and triggering, changes in protein and fatty acid metabolism, signaling, and transport pathways were most apparent. Unique proteomic fingerprints distinguish among IFN-gamma-specific, LPS-specific, or M1-specific activation states and provide a clear molecular, archeological profile to infer recent history of cells, as well as correlates for chronic macrophage activation in health and disease.

Figure 1.

Profile of LPS- and IFN-γ-induced cytokine secretion by macrophages. Macrophages were treated with media alone (mock, ○), 1 μg/ml LPS (•), or 1 μg/ml IFN-γ (shaded circles) for 1, 12, 24, and 48 h. Accumulation of (A) TNF, (B) IL-6, (C) IL-1β, and (D) CXCL10 was determined by ELISA. Data represent mean ± sd of three independent monocyte donors.

Figure 2.

Distribution of proteins across m.w. regions. Heat-gel representing proteins identified in gel slices following trypsinization and LC-MS analysis (A). Gel slices 1–15 are shown on the left with representative m.w. and P values. Heat-gels for Donors 1–3 for 24 h at a basal level, IFN-γ-primed, and LPS-activated. Gel slices are colored to represent the number of protein identifications within that mass region and scaled to reflect the relative size of the slice analyzed. Histogram of the percentage of proteins observed in each m.w. region (B). Basal, IFN-γ, and LPS are represented by blue, red, and green, respectively.

Figure 3.

Distribution of observed proteins relative to predicted molecular mass. A total of 4474 observed proteins was binned according to identified m.w. regions and plotted against their respective predicted m.w. The shaded area indicates the m.w. region that each bin covers, with largest to smallest proteins arranged from upper right to lower left, respectively. Red vertical lines represent the median values, and horizontal lines indicate the interquartile ranges for the predicted m.w.

Figure 4.

Response profiles of observed proteins. Proteins observed in two out of three donors from basal (red), IFN-γ (blue), and LPS (yellow) are shown to scale (A). The white region represents the core proteome; i.e., proteins present under all conditions. The colored regions indicate the response proteome. The table indicates the number of proteins observed for each treatment (Total) and within each region of the Venn diagram by color. The elliptical Venn diagram displays overlap among response proteomes (B).

Figure 5.

Heat-map of representative proteins expressed exclusively after 24 h of IFN-γ or LPS treatment across donors. Heat-map illustrates proteins that are expressed exclusively upon IFN-γ treatment (IFN-γ-responsive, blue), LPS treatment (LPS-responsive, yellow), and IFN-γ or LPS (M1-responsive, green). Protein symbols, Human Protein Reference Database (HPRD), SwissProt, and P value identifiers are represented on the right.

Figure 6.

Intracellular proteomic profile of IFN-γ-primed or TLR4-activated macrophages. AA=arachidonic acid, COX-2=cyclooxygenase 2, cPLA₂=cytoplasmic phospholipase A₂, GRB2=growth factor receptor-bound protein 2, IGF2R=insulin-like growth factor 2 receptor, iNOS- inducible NO synthase, iPLA₂=Ca²⁺- independent cytosolic phospholipase A₂, IRF3=IFN regulatory factor 3, Lyso-PtdCho=lysophosphotidylcholine, MRC1= mannose receptor 1, PAK2=p21-activated kinase 2, sPLA₂=secretory phospholipase A₂.