NADPH Oxidases Are Essential for Macrophage Differentiation

Abstract

NADPH oxidases (NOXs) are involved in inflammation, angiogenesis, tumor growth, and osteoclast differentiation. However, the role of NOX1 and NOX2 in macrophage differentiation and tumor progression is still elusive. Here we report that NOX1 and NOX2 are critical for the differentiation of monocytes to macrophages, the polarization of M2-type but not M1-type macrophages, and the occurrence of tumor-associated macrophages (TAMs). We found that deletion of both NOX1 and NOX2 led to a dramatic decrease in ROS production in macrophages and resulted in impaired efficiency in monocyte-to-macrophage differentiation and M2-type macrophage polarization. We further showed that NOX1 and NOX2 were critical for the activation of the MAPKs JNK and ERK during macrophage differentiation and that the deficiency of JNK and ERK activation was responsible for the failure of monocyte-to-macrophage differentiation, in turn affecting M2 macrophage polarization. Furthermore, we demonstrated that the decrease in M2 macrophages and TAMs, concomitant with the reduction of cytokine and chemokine secretion, contributed to the delay in wound healing and the inhibition of tumor growth and metastasis in NOX1/2 double knockout mice compared with WT mice. Collectively, these data provide direct evidence that NOX1 and NOX2 deficiency impairs macrophage differentiation and the occurrence of M2-type TAMs during tumor development.

Keywords: NADPH oxidase; cell biology; cell differentiation; macrophage; reactive oxygen species (ROS).

FIGURE 1.

NOX1 and NOX2 depletion inhibited ROS production and O₂^˙̄ generation in monocytes. A, the expression levels of the NOX family (NOX1, NOX2, NOX3, NOX4, Duox1, and Duox2) in mouse BMMs and BMDMs were detected by real-time PCR assay. B, the expression levels of NOX1, NOX2, NOX4, and Duox1 in BMDMs from WT, NOX1-KO, NOX2-KO, and NOX1/2-DKO mice were detected by real-time PCR assay. C, mouse BMMs from WT, NOX1-KO, NOX2-KO, and NOX1/2-DKO mice were treated with M-CSF (20 ng/ml) at the indicated time points. Cells were stained with CM-H2DCFDA for 30 min and then washed and fixed. The percentage of DCFDA-positive population was quantified by flow cytometry. D, mouse BMMs from WT and NOX1/2-DKO mice were collected. Lucigenin (200 mm) was added, and chemiluminescence was measured after treatment with or without M-CSF (20 ng/ml) over the indicated time period. Relative O₂^˙̄ generation was determined relative to the relative light units per second at the 0 time point for each group. *, p < 0.05 at all time points when WT+MCSF compared with WT baseline; #, p < 0.05 at all time points when WT+MCSF compared with NOX1/2-DKO+MCSF; **, p < 0.05 at most time points when NOX1/2-DKO baseline compared with WT baseline.

FIGURE 2.

Absence of both NOX1 and NOX2 affects monocyte-to-macrophage differentiation. A, BMMs from C57BL/6 WT, NOX1-KO, NOX2-KO, and NOX1/2-DKO mice were treated with M-CSF (20 ng/ml) for 6 days (M0). On day 6, the M0 cells were treated with LPS (100 ng/ml) and INF-γ (20 ng/ml) for M1 or IL-4 (20 ng/ml) for M2 for 24 h. Representative light microscopy image from three independent experiments are shown. B, cells from A were analyzed by flow cytometry with antibodies to iNOS and F4/80 (M1 population) and to RELMα and F4/80 (M2 population). C, detection of M1 cytokines (TNF-α, ΙL-12β) and M2 chemokines (CCL17, CCL24) by real-time PCR of cells from A. D and E, mouse peritoneal macrophages from WT and NOX1/2-DKO were isolated and cultured overnight. Then macrophages were polarized to M1 or M2 macrophages. Cells were co-stained with the indicated antibodies and analyzed by flow cytometry (D). The expression levels of M1 cytokines (TNF-α, IL-12β) or M2 chemokines (CCL17, CCL24) were detected by real-time PCR (E). F and G, WT, NOX1-KO, NOX2-KO, and NOX1/2-DKO mice (n = 3/group) were injected i.p. with either TG or IL-4c. On day 4 after injection, macrophages were isolated by peritoneal lavage, co-stained with the indicated antibodies, and analyzed by flow cytometry (F). The expression levels of M1 cytokines (TNF-α, IL-12β) or M2 chemokines (CCL17, CCL24) were detected by real-time PCR (G). All results represent the mean ± S.D. from three independent experiments. *, p < 0.05 versus WT.

FIGURE 3.

NOX1 and NOX2 depletion inhibited ROS-mediated ERK and JNK activation. A, mouse BMMs from WT, NOX1-KO, NOX2-KO, and NOX1/2-DKO mice were treated with M-CSF (20 ng/ml) for the indicated time points. The expression levels of p-ERK, ERK, p-JNK, and JNK were determined by Western blotting. B, mouse monocytes from WT, NOX1-KO, NOX2-KO, and NOX1/2-DKO mice were treated with M-CSF for 6 days. On day 6, cells were treated with LPS/INF-γ (left panel) or IL-4 (right panel) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. C, on day 0, mouse monocytes from WT, NOX1-KO, NOX2-KO, and NOX1/2-DKO mice were treated with DMSO or ERK inhibitor (U0126, 5 μm) for 1 h and then treated with M-SCF for 6 days. On day 6, the cells were analyzed by imaging and flow cytometry. Representative light microscopy image are shown (top panel). Macrophages were collected and stained with anti-F4/80 antibody and analyzed by flow cytometry (bottom panel). D and E, on day 6 the macrophages from C were further treated for 24 h with either LPS (100 ng/ml) and INF-γ (20 ng/ml) for M1 or IL-4 (20 ng/ml) for M2 polarization. The M1 cell population (iNOS and F4/80) and M2 cell population (RELMα and F4/80) were detected by flow cytometry (D). Detection of M1 cytokines (TNF-α, IL-12β) or M2 chemokines (CCL17, CCL24) was by real-time PCR (E). *, p < 0.05; **, p < 0.001 versus WT.

FIGURE 4.

NOX1 and NOX2 depletion has no effect on macrophage pro-inflammatory function. A and B, measuring production of IL-1β (A) or Western blotting analysis of expression levels of Caspase1 and Caspase1p20 (B) of LPS-primed BMDMs treated with 5 mm ATP (30 min) or 20 μm nigericin (2 h), transfected with poly(A):T (6 h), or infected with S. Typhimurium (S. typhi, 10⁸ cfu for 6 h).

FIGURE 5.

Wounding healing is delayed in NOX1 and NOX2 double knockout mice. A, analysis of skin wound healing over time. One representative image from two independent experiments with four animals per time point for each group is shown. B, percentage of wound area at each time point relative to the original wound area for mice from A. C, immunofluorescence staining of Arginase 1 (red), iNOS (green), and nucleus (blue) at wound sites from WT and NOX1/2-DKO mice at the indicated times. Representative images are shown. D, the expression levels of mouse IL-1β, TNF-α, iNOS, Arginase 1, TGF-β, VEGF, and IL-10 at the wound sites were analyzed by real-time PCR at the indicated time points. *, p < 0.05 versus WT.

FIGURE 6.

NOX1 and NOX2 deficiency inhibits LLC tumor growth and metastasis. A–C, LLC cells were subcutaneously injected into WT and NOX1/2-DKO mice. Xenografts were removed 33 days after implantation, and tumors were photographed. Representative images are shown (A). Tumor weights at 33 days (B) and tumor volumes for the indicated times (C) were obtained and are presented (mean ± S.E., n = 8/group). The p values are indicated. D, the percentage of F4/80+ cells in primary tumors on day 33 was determined by flow cytometry. Shown is quantification of the percentage of RELMα+ cells in the F4/80+ population. *, p < 0.05. E, representative immunofluorescence images showing Arginase I (red), F4/80 (green), and nucleus (blue) staining of primary tumors from WT and NOX1/2-DKO mice on day 33. F, the expression levels of TNF-α, iNOS, Arginase 1, CCL24, VEGF, and IL-10 in lymphocytes isolated from day 33 tumors from WT and NOX1/2-DKO mice were analyzed by real-time PCR. * p < 0.05. G, representative images of H&E staining of lung sections 33 days after LLC cell injection (top panel). Whole lung sections were scanned and scored for metastatic foci (bottom panel). Metastasis index = metastasis number divided by primary tumor weight (mean ± S.E., n = 8/group).