IL-25 Induced ROS-Mediated M2 Macrophage Polarization via AMPK-Associated Mitophagy

Abstract

Interleukin (IL)-25 is a cytokine released by airway epithelial cells responding to pathogens. Excessive production of reactive oxygen species (ROS) leads to airway inflammation and remodeling in asthma. Mitochondria are the major source of ROS. After stress, defective mitochondria often undergo selective degradation, known as mitophagy. In this study, we examined the effects of IL-25 on ROS production and mitophagy and investigated the underlying mechanisms. The human monocyte cell line was pretreated with IL-25 at different time points. ROS production was measured by flow cytometry. The involvement of mitochondrial activity in the effects of IL-25 on ROS production and subsequent mitophagy was evaluated by enzyme-linked immunosorbent assay, Western blotting, and confocal microscopy. IL-25 stimulation alone induced ROS production and was suppressed by N-acetylcysteine, vitamin C, antimycin A, and MitoTEMPO. The activity of mitochondrial complex I and complex II/III and the levels of p-AMPK and the mitophagy-related proteins were increased by IL-25 stimulation. The CCL-22 secretion was increased by IL-25 stimulation and suppressed by mitophagy inhibitor treatment and PINK1 knockdown. The Th2-like cytokine IL-25 can induce ROS production, increase mitochondrial respiratory chain complex activity, subsequently activate AMPK, and induce mitophagy to stimulate M2 macrophage polarization in monocytes.

Keywords: AMPK; IL-25; M2 macrophage polarization; mitophagy; reactive oxygen species.

Figure 1

IL-25 alone could induce intracellular ROS level and suppressed by NAC, vitamin C, antimycin A and MitoTEMPO. (A–E), representative histograms of cell counts (counts) vs. DCF fluorescence (FITC-A) (above), and the mean fluorescence intensity (MFI) of DCF is expressed as percentage relative to control cells (below). THP-1 cells (white bar) and THP-1 derived macrophages (gray bar) treated with medium alone or IL-25 (2, 10 and 40 ng/mL) was for 0.5 h (A) or 2 h (B). (C) Cells were treated with medium alone or IL-25 (40 ng/mL) present pretreatment with NAC (1 and 5 mM) or vitamin C (1 and 10 μM) for 0.5 h. After pretreatment with solvent control (0.1% DMSO) or antimycin A (0.1 and 0.5 μg/mL) (D), or medium alone or MitoTEMPO (10 and 100 μM) (E) for 0.5 h followed by IL-25 treatment, the ROS level was measurement. * p < 0.05, ** p < 0.01, and *** p < 0.001; n = 3, means ± SD.

Figure 2

IL-25 increased mitochondria complex activity in THP-1 cells and THP-1 derived macrophages. THP-1 cells (white bar) and THP-1 derived macrophages (gray bar) were treated with medium alone or IL-25 (2, 10, and 40 ng/mL) for 2 h. The activity of complex I (A) and complex II/III (B) by IL-25 treatment were determined. * p < 0.05, ** p < 0.01, and *** p < 0.001; n = 4, means ± SD.

Figure 3

IL-25-induced AMPK activation and mitophagy-related proteins expression in THP-1 cells and THP-1 derived macrophages. (A) The time course of p-AMPK expression was determined at 1, 3, 6, 8, and 12 h with IL-25 (40 ng/mL) or medium alone treatment in THP-1 cells (white bar) and THP-1 derived macrophages (gray bar). Data present the means ± SD of 3 independent experiments. The expression of p-AMPK (B), PINK1 (C), p-parkin (D), and LC3 (E) by medium alone or IL-25 (2, 10, and 40 ng/mL) treatment in THP-1 cells (white bar) and THP-1 derived macrophages (gray bar) at 8 h time point were determined by western blot. * p < 0.05, ** p < 0.01, and *** p < 0.001; n = 3, means ± SD. The confocal data show the nuclear (DAPI), mitochondria (MitoTracker), and LC3 I/II fluorescence images by medium alone or IL-25 (10 ng/mL) treatment (F). Scale bars = 10 μm in pictures.

Figure 4

IL-25-induced mitophagy-related proteins expression via ROS-AMPK pathway in THP-1 cells and THP-1 derived macrophages. (A) representative histograms of cell counts (counts) vs. DCF fluorescence (FITC-A) (above), and the mean fluorescence intensity (MFI) of DCF expressed as a percentage relative to control cells (below). (A) After pretreatment with solvent control (0.1% DMSO) or AMPKi (1 and 10 µM) for 0.5 h, the IL-25-induced ROS production in THP-1 cells (white bar) and THP-1 derived macrophages (gray bar) were measured. n = 3, means ± SD. (B) After medium alone or NAC (1 and 5 mM) pretreatment for 0.5 h followed by IL-25 treatment for 8 h, IL-25-induced p-AMPK expression in THP-1 cells (white bar) and THP-1 derived macrophages (gray bar) were determined. n = 4, means ± SD. (C) After pretreatment with solvent control (0.1% DMSO), rotenone (1 μM) or antimycin A (0.5 μg/mL) for 0.5 h followed by IL-25 treatment for 8 h, IL-25-induced p-AMPK expression in THP-1 cells (white bar) and THP-1 derived macrophages (gray bar) were deter-mined. n = 3, means ± SD. After pretreatment with solvent control (0.1% DMSO) or AMPKi (1 and 10 µM) for 0.5 h followed by IL-25 treatment for 8 h, the PINK1 (D), p-Parkin (E), and LC3 I/II (F) protein levels were determined by western blot. n = 3, means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 5

IL-25-induced mitophagy via PINK1-Parkin pathway in THP-1 cells and THP-1 derived macrophages. (A,F) representative histograms of cell counts (counts) vs. DCF fluorescence (FITC-A) (above), and the mean fluorescence intensity (MFI) of DCF expressed as a percentage relative to control cells (below). (A) After pretreat-ment with solvent control (0.1% DMSO) or mdivi-1 for 0.5 h, the IL-25-induced ROS production in THP-1 cells (white bar) and THP-1 derived macrophages (gray bar) were measured by flow cytometry. n = 3, means ± SD. After pretreatment with solvent control (0.1% DMSO) or mdivi-1 for 0.5 h followed by IL-25 treatment for 8 h, the PINK1 (B), p-Parkin (C), and LC3 I/II (D) protein levels of THP-1 cells (white bar) and THP-1 derived macrophages (gray bar) were determined. n = 3, means ± SD. (E) After transduction with shRNA lentiviral particles and puromycin selection for 3 d, the PINK1 expression was determined. (F) After PINK1 knockdown by TRCN0000007097 or TRCN0000199446 shRNA, the ROS level by medium alone or IL-25 (2, 10, and 40 ng/mL) treatment in THP-1 cells at 2 h was measured. n = 3, means ± SD. The expression of PINK1 (G), p-Parkin (H), LC3 I/II (I) by medium alone or IL-25 (40 ng/mL) treatment following transduction with PINK1 shRNA or non-targeting control were determined. n = 3, means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

IL-25-induced mitophagy was associated with a shift in M2 macrophage polarization. THP-1 cells were pretreated with medium alone or IL-25 (2, 10, and 40 ng/mL) for 2 h and differentiated to THP-1 derived macrophages (PMA), M1 (PMA/LPS/IFN-γ), or M2 (PMA/IL-4) macrophages. The concentration of M1-related chemokine CXCL-10 (A) and cy-tokine TNF-α (B) and M2-related cytokine CCL-22 (C) were measured. After pretreatment with solvent control (0.1% DMSO) or mdivi-1 (D), or knockdown PINK1 (E), the IL-25-induced CCL-22 production was assessed. * p < 0.05, ** p < 0.01, and *** p < 0.001; n = 3, means ± SD.

Figure 7

Schematic of the proposed intracellular mechanisms underlying IL-25-induced M2 macrophage polarization. IL-25 increased ROS production and mitochondrial complex I and complex II/III activity. Subsequently, activation of mitophagy, and then shifted M1/M2 chemokine expression via the AMPK signaling pathway in human monocyte cell line.