Angiotensin II modulates THP-1-like macrophage phenotype and inflammatory signatures via angiotensin II type 1 receptor

Abstract

Angiotensin II (Ang II) is a major component of the renin-angiotensin or renin-angiotensin-aldosterone system, which is the main element found to be involved in cardiopathology. Recently, long-term metabolomics studies have linked high levels of angiotensin plasma to inflammatory conditions such as coronary heart disease, obesity, and type 2 diabetes. Monocyte/macrophage cellular function and phenotype orchestrate the inflammatory response in various pathological conditions, most notably cardiometabolic disease. An activation of the Ang II system is usually associated with inflammation and cardiovascular disease; however, the direct effect on monocyte/macrophages has still not been well elucidated. Herein, we have evaluated the cellular effects of Ang II on THP-1-derived macrophages. Ang II stimulated the expression of markers involved in monocyte/macrophage cell differentiation (e.g., CD116), as well as adhesion, cell-cell interaction, chemotaxis, and phagocytosis (CD15, CD44, CD33, and CD49F). Yet, Ang II increased the expression of proinflammatory markers (HLA-DR, TNF-α, CD64, CD11c, and CD38) and decreased CD206 (mannose receptor), an M2 marker. Moreover, Ang II induced cytosolic calcium overload, increased reactive oxygen species, and arrested cells in the G1 phase. Most of these effects were induced via the angiotensin II type 1 receptor (AT1R). Collectively, our results provide new evidence in support of the effect of Ang II in inflammation associated with cardiometabolic diseases.

Keywords: ROS; angiotensin II; angiotensin II type 1 receptor (AT1R); inflammation; macrophages.

Figure 1

Ang II change THP-1-like macrophage phenotype. The number of positive cells in frequency (%) of (A) CD116, (B) CD64, (C) CD38, (D) HLA-DR, (E,F) CD11c, (G) TNF-α, and (H) CD206 were analyzed by flow cytometry using THP-1-derived macrophage treated with angiotensin II (Ang II) (1 µM) for 24 h or untreated cells (Control). In a separate experiment, and before the Ang II treatment, the cells were incubated with Losartan (100 µM) for 2 h (Ang II/Los) (F). The results are presented as means ± SEM, n = 3–4. *P < 0.05 and **P < 0.01 and compared with controls.

Figure 2

Ang II induces apoptosis and cell cycle arrest. THP-1 macrophages were treated with angiotensin II (Ang II) (1 µM) for 24 h or untreated cells (Control). Apoptosis was measured using Annexin V staining. (A) Annexin V positive cells after exposure to Ang II. (B) Separate experiment for apoptotic level in cells treated with Ang II (1 µM) for 24 h or Losartan (100 µM) and Ang II (Ang II/Los). (C) Cell cycle analysis showing G1, S, and G2 phases. All samples were analyzed using flow cytometry (BD LSR Fortessa). The results are presented as means ± SEM, n = 3–4. *P < 0.05 and **P < 0.01 and compared with controls.

Figure 3

Ang-II-induced ROS production. THP-1 macrophages were treated with angiotensin II (Ang II) (1 µM) for 24 h; Ang II and Losartan (Ang II/Los) (100 µM) or untreated cells (Control). Confluent cells prepared in suspensions at 10⁵–10⁶ cells/ml were incubated with CellROX™ Deep Red reagent (5 μm) for 30 min at 37°C, 5% CO₂, protected from light, then analyzed using flow cytometry (BD LSR Fortessa). (A) ROS production (% of control) in cells treated with Ang II and (B) ROS production in a separate experiment for cells treated with Losartan and Ang II. The results are presented as means ± SEM, n = 3–4. *P < 0.05 and **P < 0.01 and compared with controls.

Figure 4

Ang II increases intracellular calcium and fluorescence recovery after photobleaching (FRAP). Cell surface topography during cell elevation of cytosolic Ca²⁺ and change of FRAP signal. The images show fluo4 staining for intracellular calcium, Dil image (Dil) for peripheral cell location, and the phase contrast image (PC). (A) Cell surface topography for THP-1 macrophages untreated cells (Control) and (B) cells treated with angiotensin II (Ang II) (1 µM) for 24 h. The cells were loaded with the Fluo4 Ca²⁺ indicator, and Dil. After the cell was loaded with calcium indicator Flou4/AM, the cells were tested for FRAP intensity before exposure to UV light (first bleach) and after changing (second bleach). Exposure to UV light by using transient illumination with 404 nm laser. The graphs present the Fluo4 intensity and the time courses of Dil fluorescence. (D) and (E) Fluo4 fluorescence corresponding to cytosolic free Ca²⁺ measured as median fluorescent intensity. The results are presented as means ± SEM, n = 3. *P < 0.05 and **P < 0.01 and compared with controls.

Figure 5

Ang II increases monocyte adhesion to HUVEC cells. The number of positive cells in frequency (%) of (A) CD49F, (B) CD33, (C) CD15, and (D) CD44 were analyzed by flow cytometry using THP-1-derived macrophage cells treated with Ang II (1 µM) for 24 h; Losartan (100 µM) and Ang II (Ang II/Los) or untreated cells (Control). (E,F) The number of adherent monocytes onto HUVECs, determined using EVOS microscope. The results are presented as means ± SEM, n = 4. *P < 0.05 and **P < 0.01 and compared with controls.

Figure 6

Ang II increases the phosphorylation level of kinases in THP-1 macrophages. Analysis of a proteome profiler for phospho-kinases using THP-1-derived macrophage cells treated with Ang II (1 µM) for 24 h; Losartan (100 µM) for 2 h before the Ang II treatment (Ang II/Los) or untreated cells (Control). Phosphorylated signaling proteins presented as densitometric analysis. (A) Selected proteins inhibited by Losartan. (B) Data of each array (Part A and B) incubated with 100 μg of cell lysate, shown from a 5-min exposure. (C) Proteins panel differentially expressed following the Ang II treatment without significant effect of Losartan. The results are presented as means ± SEM, *P < 0.05, and **P < 0.01 and compared with controls.

Figure 7

Graphical summary suggested pathways that might be implicated at least in part in angiotensin II inducing monocyte/macrophage dysfunction and polarization (red arrow) and angiotensin type 1 receptor involvement in this process (green arrow). Los, Losartan; ROS, reactive oxygen species; Ca²⁺, intracellular calcium. CD markers for cell phenotype and macrophage polarization: CD116, CD64, CD38, HLA-DR, CD15, CD33, CD49F, CD44, CD11c, TNF-α, and CD206. Human kinases: Akt 1/2/3, GSK-3 alpha/beta, Chk-2 (T68), RSK1/2/3 (S380), ERK1/2 (T202/Y204, T185/Y187), STAT3, p38 alpha, eNOS, STAT1, STAT2, MSK1/2, RSK1/2/3, HSP27 (S78/S82), Lyn (Y397), PRAS40 (T246), and HSP60.

Figure 8

Schematic presentation of potential pathways associated with angiotensin -II-induced macrophages proinflammatory-like phenotype polarization. Angiotensin II (Ang II) stimulate reactive oxygen species (ROS) and heat shock protein 60 (HSP60) via angiotensin II type 1 receptor (AT1R) to stimulate p38a/ERK1/2 pathways, which promote macrophage proinflammatory-like phenotype polarization directly by interleukin 6 (IL-6) release or through RSK1/2 and STAT1/2 phosphorylation to increase tumor necrosis factor alpha (TNF-α) production. These effects are potentially inhibited at least in part by Losartan (Los) treatment.