Macrophages and their precursors, monocytes, play an important role during organ injury and repair. The subset of Ly-6Chi
monocytes was increased dramatically in lesions of infection and injury in a variety of inflammatory diseases (2
). The Ly-6Chi
monocytes migrated to the inflamed/necrotic area and subsequently differentiated into mature macrophages. This process required downregulation of Ly-6C expression and an increase in the expression of the mature macrophage marker F4/80 (2
). In the present study, we demonstrated that the loss of Foxm1 in a myeloid lineage reduced numbers of Ly-6Clo
mature macrophages at 72 h after liver injury, coinciding with delayed liver repair. The numbers of monocytic macrophage precursor cells were reduced in mFoxm1−/−
livers 48 h after liver injury, a time prior to the accumulation of mature macrophages in the liver. These results suggest that Foxm1 may play an important role in liver repair by inducing migration of Ly-6Chi
monocytes into the injured liver. Consistent with this hypothesis, we found decreased expression of L-selectin in Foxm1-deficient monocytes. L-selectin is a cell adhesion molecule essential for monocyte migration (25
). Alternatively, reduced numbers of mature macrophages in injured mFoxm1−/−
livers can be a direct consequence of impaired differentiation of monocytes toward the macrophage cell lineage. Interestingly, we found no differences in the total numbers of neutrophils that migrated into an injured liver in mFoxm1−/−
mice. Furthermore, Foxm1 did not influence cell surface expression of L-selectin in these cells. These results suggest that Foxm1 is dispensable for neutrophil recruitment to the injured liver.
In the present study, expression of CCR2 was decreased in mFoxm1−/−
monocytes. Previous studies demonstrated that CCR2−/−
mice exhibited decreased macrophage infiltration following acetaminophen-induced liver injury, indicating that MCP-1/CCR2 signaling is essential for monocyte migration to the injured liver (9
). CCR2 gene expression is regulated by several signaling pathways, including calcineurin/NFAT (13
), IFN-γ/Stat1 (10
), and PPARγ (3
). Liver repair was delayed following acetaminophen-mediated liver injury in CCR2−/−
). Therefore, decreased CCR2 expression in mFoxm1−/−
monocytes can contribute to impaired monocyte recruitment, causing a delay in liver repair in mFoxm1−/−
mice. The finding that Foxm1 directly stimulated transcriptional activity of the CCR2 promoter and that this stimulation required the Foxm1 binding site located between the bp −269 and +22 of the mouse CCR2 promoter provide a potential mechanism by which Foxm1 influences CCR2 levels in monocytes. Our studies suggest that Foxm1 is a critical transcriptional activator of the CCR2 gene during liver injury and repair.
Previous studies focused on the role of Foxm1 protein in hepatocytes, the major cell population in the liver. Liver regeneration and liver injury studies with Alb-Cre Foxm1−/−
mice demonstrated that Foxm1
is essential for DNA replication and mitosis in hepatocytes by altering expression of proteins that are required for cell cycle progression (41
). Consistent with these studies, a majority of Foxm1−/−
embryos died in utero
by E16.5 due to a 75% reduction in the number of hepatoblasts because they failed to progress into mitosis causing a polyploid phenotype (26
). We demonstrated here that mFoxm1−/−
livers displayed a specific Foxm1 deletion in myeloid cells but not in hepatocytes. However, the proliferation of both hepatocytes and nonhepatocytes was decreased in CCl4
livers. It is possible that Foxm1 influences the secretion of inflammatory mediators, which in turn indirectly affects proliferative responses and liver repair after CCl4
injury. We also found that adoptive transfer of Foxm1fl/fl
monocytes to CCl4
mice restored liver repair and rescued liver function after the injury. These results demonstrated a critical role of Foxm1 in monocytes and established a direct link between monocyte migration and liver repair after the injury.
Numbers of cells in the G0
, S, and G2
/M phases of the cell cycle among all three major populations of hepatic myeloid cells (neutrophils, monocytes, and macrophages) were not influenced by Foxm1 deletion. Considering the important role of Foxm1 in cell cycle regulation of hepatocytes, fibroblasts, endothelial cells, and numerous neoplastic cells (18
), these results are surprising and indicate that Foxm1 is not required for proliferation of myeloid cells during either normal hematopoiesis or acute liver injury. Interestingly, recent studies showed that a conditional Foxm1 deletion from respiratory epithelial cells did not alter proliferation of these cells during lung development but caused impaired lung maturation and decreased surfactant production (16
). Altogether, these studies indicate that Foxm1 plays distinct roles in different cell populations in vivo
. Although the molecular mechanisms of this Foxm1 selectivity are still unknown, various cell-specific transcription factors may function as coactivators with Foxm1, altering a set of Foxm1 transcriptional targets and influencing cellular responses to Foxm1. Alternatively, it is possible that other cellular pathways compensate for Foxm1 deficiency in myeloid inflammatory cells.
In summary, deletion of Foxm1 in myeloid cells caused a delay in liver repair and increased pericentral apoptosis following CCl4-mediated injury. Although the recruitment of neutrophils was not influenced by the Foxm1 deficiency, reduced numbers of infiltrating macrophages were found in mFoxm1−/− livers after injury. Monocyte recruitment during liver repair was dependent upon Foxm1 expression in myeloid cells, a finding that may be related to reduced expression of L-selectin and CCR2 chemokine receptor. Foxm1 is critical for transcriptional activation of the mouse CCR2 promoter and required for the recruitment of monocytes to the injured liver.