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ROS is Required for Alternatively Activated Macrophage Differentiation

BHA blocks GM-CSF or M-CSF- induced ROS generation and the second phase of ERK activation, and further blocks the polarization to M2 macrophages. The effect of BHA is specific for M2 cells, since the inhibition effect of BHA is overcome during M1 but not M2 polarization. Tumor-associated macrophages (TAMs), which are responsible for tumor-promoting activities, are alternatively activated, or M2-like macrophages. Blocking the function of M2 cells/TAMs inhibits tumorigenesis.
BHA blocks GM-CSF or M-CSF- induced ROS generation and the second phase of ERK activation, and further blocks the polarization to M2 macrophages. The effect of BHA is specific for M2 cells, since the inhibition effect of BHA is overcome during M1 but not M2 polarization. Tumor-associated macrophages (TAMs), which are responsible for tumor-promoting activities, are alternatively activated, or M2-like macrophages. Blocking the function of M2 cells/TAMs inhibits tumorigenesis.

Macrophages are key regulators in host inflammatory responses. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are responsible for inducing macrophage differentiation from monocytes. GM-CSF or M-CSF-differentiated macrophages can be further differentiated, or polarized, to more specialized cells. Classically activated, or M1, macrophages have immune-stimulatory properties and cytotoxic function against tumor cells. Alternatively activated, or M2, macrophages have low cytotoxic function but high tissue-remodeling activity. There are also M2-like cells called tumor-associated macrophages (TAMs) that are responsible for many tumor-promoting activities. Blocking the function of TAMs inhibits tumorigenesis.

When there is loss of regulation of macrophage differentiation, diseases such as autoimmune disorders and cancer can result. The underlying mechanisms of macrophage differentiation remain unclear, but some studies have suggested that reactive oxygen species (ROS) may be involved. Zheng-Gang Liu, Ph.D., and his colleagues in CCR’s Cell and Cancer Biology Branch set out to investigate the role of ROS in macrophage differentiation.

The researchers began their studies by investigating whether necrosis is involved in monocyte death. They tested the effect of butylated hydroxyanisole (BHA), which inhibits necrosis in some cells, on the death of human primary monocytes in cell cultures. BHA resulted in the loss of GM-CSF- and M-CSF-induced macrophage morphologies, suggesting that BHA may affect monocyte-macrophage differentiation. They then examined whether BHA affects the differentiation of M1 and M2 macrophages. BHA specifically blocked differentiation of human monocytes to M2 but not M1 macrophages.

BHA is a known ROS-inhibitor, so the researchers investigated whether BHA affected M2 macrophage differentiation via ROS elimination. Treatment with BHA inhibited GM-CSF- and M-CSF-induced superoxide production. Additional experiments demonstrated that ROS plays a key role in the differentiation of M2 macrophages, and BHA blocks differentiation of M2 macrophages by inhibiting NADPH oxidase-mediated superoxide production, which is the main non-mitochondrial source of ROS.  

Three mitogen-activated protein kinases (MAPKs), ­ERK, JNK, and p38, are activated following GM-CSF or M-CSF treatment and are involved in monocyte-macrophage differentiation. To determine the mechanism of ROS-mediated macrophage differentiation, the researchers examined which MAPK pathway is required for human primary monocyte differentiation using inhibitors specific for each pathway. GM-CSF- or M-CSF-induced ROS generation was required for late-phase ERK activation, which is needed for monocyte-macrophage differentiation. Further experiments indicated that blocking ERK activation in the early stage of macrophage differentiation specifically affects the M2 differentiation, and loss of ERK activation in GM-CSF-differentiated macrophages is overcome during M1 polarization. In addition to using human monocytes, the researchers carried out in vitro and in vivo studies on murine macrophages and found that the requirement of ROS in the differentiation of monocytes to M2 macrophages is a general phenomenon.

Since M2 macrophages share many features with TAMs, whether BHA blocks the occurrence of TAMs in three mouse tumor models was investigated. The first model that was used was a Urethane-induced lung cancer model in which TAMs are thought to play a role in tumorigenesis. Continuous administration of BHA blocked the occurrence of TAMs and suppressed tumorigenesis at both initiation and progression stages. BHA also inhibited the occurrence of TAMs and suppressed the development of primary tumors in the K-ras-induced lung cancer and MMTV-PyMT-induced breast cancer models.

To determine whether BHA affected monocyte recruitment, MCP-1, which is the major chemokine regulating monocyte recruitment, was used in a monocyte transwell migration assay. MCP-1 increased cellular migration of human monocytes, and the presence of BHA had no effect on MCP-1-induced monocyte migration.

To further demonstrate that BHA suppresses tumorigenesis by blocking M2 macrophage/TAM differentiation, the effect of BHA on tumor cell growth in vitro and in vivo was examined. The presence of BHA had no effect on the proliferation of three types of tumor cell lines but did reduce tumor growth in a breast cancer xenograft model. When macrophages were depleted, the presence of BHA did not suppress tumor growth.

Taken together, the results suggest that BHA blocks tumor growth by specifically affecting TAMs but not the proliferation of tumor cells directly. This study demonstrates that continuous administration of ROS inhibitors for targeting TAMs may be a potentially effective method for cancer treatment.

Reference
Zhang Y, Choksi S, Chen K, Pobezinskaya Y, Linnoila I, Liu ZG. ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages. Cell Research. July 2013. PubMed Link

Reviewed by Miranda L Hanson