Hypoxia modifies the polarization of macrophages and their inflammatory microenvironment, and inhibits malignant behavior in cancer cells

Abstract

Macrophages are a heterogeneous group of phagocytes that play critical roles in inflammation, infection and tumor growth. Macrophages respond to different environmental factors and are thereby polarized into specialized functional subsets. Although hypoxia is an important environmental factor, its impact on human macrophage polarization and subsequent modification of the inflammatory microenvironment have not been fully established. The present study aimed to elucidate the effect of hypoxia exposure on the ability of human macrophages to polarize into the classically activated (pro-inflammatory) M1, and the alternatively activated (anti-inflammatory) M2 phenotypes. The effect on the inflammatory microenvironment and the subsequent modification of A549 lung carcinoma cells was also investigated. The presented data show that hypoxia promoted macrophage polarization towards the M2 phenotype, and modified the inflammatory microenvironment by decreasing the release of proinflammatory cytokines. Modification of the microenvironment by proinflammatory M1 macrophages under hypoxia reversed the inhibition of malignant behaviors within the proinflammatory microenvironment. Furthermore, it was identified p38 signaling (a major contributor to the response to reactive oxygen species generated by hypoxic stress), but not hypoxia-induced factor, as a key regulator of macrophages under hypoxia. Taken together, the data suggest that hypoxia affects the inflammatory microenvironment by modifying the polarization of macrophages, and thus, reversing the inhibitory effects of a proinflammatory microenvironment on the malignant behaviors of several types of cancer cell.

Keywords: hypoxia; hypoxia-induced factor-1α; macrophage; malignant behavior; p38.

Figure 1.

Identification of M1 and M2 macrophage phenotypes after differentiation. (A) mRNA levels of IL-1β, TNF-α, VEGF, HLA-DR, CCL17 and CD163 in MΦ, M1 and M2 macrophages after specific stimulation. *P<0.05, vs. Mφ-stimulated group; ^#P<0.05, vs. Mφ-stimulated group. (B) Protein levels of IL-1β, TNF-α, HLA-DR, CCL17 and CD163 in MΦ-, M1- and M2-polarized macrophages. *P<0.05, vs. Mφ-stimulated group; ^#P<0.05, vs. Mφ-stimulated group. (C) CD86 and CD206 expression rates were measured. *P<0.05, vs. Mφ-stimulated group; ^#P<0.05, vs. Mφ-stimulated group. IL-1β, interleukin-β; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor; HLA-DR, human leukocyte antigen-DR; CCL17, chemokine (C-C motif) ligand 17.

Figure 2.

Effects of hypoxia on the expression levels of cytokines and chemokines. (A) mRNA levels of IL-1β, TNF-α, VEGF, HLA-DR, CCL17 and CD163 in MΦ, M1 (LPS+IFN-γ) and M2 (IL-4) macrophages after specific stimulation under normoxic or hypoxic conditions. *P<0.05, vs. Normoxic group. (B) Protein levels of IL-1β, TNF-, HLA-DR, CCL17 and CD163 in MΦ-, M1- and M2-polarized macrophages under normoxic or hypoxic conditions. *P<0.05, vs. LPS+IFN-γ/Normoxic group. (C) CD86 and CD206 expression levels. *P<0.05, vs. LPS+IFN-γ/Normoxic group. (D) Secretion of IL-1β, TNF-α, VEGF, and CCL17. *P<0.05, vs. LPS+IFN-γ/Normoxic group. IL-1β, interleukin-β; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor; CCL17, chemokine (C-C motif) ligand 17; LPS, lipopolysaccharide; IFN-γ, interferon-γ.

Figure 3.

Effects of hypoxia-modified M1-polarized conditioned medium on malignant behaviors of cancer cells. (A) Proliferation of A549, HeLa, HepG2 and MCF-7 cells was analyzed by performing CCK-8 assay. The effects of hypoxia conditioned medium on (B) Colony formation and (C) Soft agar tumor formation. *P<0.05 vs. LPS+IFN-γ/Normoxia. PMA, phorbol 12-myristate 13-acetate; CCK-8, cell counting kit-8; LPS, lipopolysaccharide; IFN-γ, interferon-γ.

Figure 4.

Hypoxia affects the inflammatory microenvironment in a p38-dependent and HIF-1α-independent manner. HIF-1α protein expression level was measured following (A) hypoxia and (B) CoCl₂ treatment. (C) mRNA levels of IL-1β, TNF-α, VEGF, HLA-DR, CCL17 and CD163 were measured after hypoxic exposure or CoCl₂ treatment. *P<0.05, vs. Normoxia-exposed group. (D) Release of IL-1β, TNF-α, VEGF and CCL17 in supernatant was measured by ELISA. (E) CD86 and CD206 expression was also assessed. *P<0.05, vs. Normoxia-exposed group. (F) p38 and phosphorylated p38 levels were measured under hypoxic conditions with or without 1 µM SB203580. (G) IL-1β, TNF-α, VEGF and CCL17 in the supernatant was measured by ELISA under hypoxic conditions with or without 1 µM SB203580. *P<0.05, vs. Normoxia-exposed group; ^#P<0.05, vs. Hypoxia-exposed group. HIF, hypoxia inducible factor; 1β, interleukin-β; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor; HLA-DR, human leukocyte antigen-DR; CCL17, chemokine (C-C motif) ligand 17; p-, phosphorylated; LPS, lipopolysaccharide; IFN-γ, interferon-γ.