Impaired DNA repair in mouse monocytes compared to macrophages and precursors

Abstract

Previously we showed that human monocytes isolated from peripheral blood display downregulation of several DNA repair proteins, including XRCC1, ligase III, PARP-1 and DNA-PK_CS, resulting in a deficiency of DNA repair, while in macrophages derived from monocytes the repair protein expression and DNA repair is restored. To see whether this is a specific phenomenon of human monocytes and macrophages, we assessed the expression of these repair genes in mice. We also addressed the question at which differentiation step in bone marrow cells downregulation of DNA repair gene expression occurs. The study revealed that mouse monocytes, similar to human, lack the expression of XRCC1, ligase III, PARP-1 and DNA-PK_CS. If mice were treated with total body irradiation, they showed significant apoptosis in bone marrow monocytes, but not in peritoneal macrophages. This was also observed after treatment with the methylating anticancer drug temozolomide, resulting in high death rate of monocytes, but not macrophages. Monocytes arise from hematopoietic stem cells. Even the early stem cell fraction (LT-HSC) expressed detectable amounts of XRCC1, which was transiently upregulated, achieving the highest expression level in CMP (common myeloid progenitor) and, during the subsequent differentiation process, downregulated up to a non-detectable level in monocytes. The immediate monocyte precursor GMP also expressed ligase III, PARP-1 and DNA-PK_CS. All these repair genes lacking in monocytes were upregulated again in macrophages. The sensitivity of monocytes, macrophages and precursor cells roughly correlated with their XRCC1 expression level. Monocytes, but not macrophages, also displayed strong γH2AX focal staining, indicating the presence of non-repaired DNA double-strand breaks following total body irradiation. Overall, the data revealed that murine monocytes exhibit the same DNA repair-impaired phenotype and high sensitivity compared to macrophages as observed in human. Therefore, the repair deficiency previously described for human monocytes appears to be a general property of this cell type.

Introduction

Monocytes and macrophages are mononuclear phagocytes that play a crucial role in tissue homeostasis and immunity. Monocytes originate from myeloid precursor cells in primary lymphoid organs, including bone marrow and fetal liver, during embryonic and adult haematopoiesis [1]. Monocytes traffic via the bloodstream to peripheral tissue where they migrate and, depending on the local growth factors, cytokines and microbial molecules, differentiate into macrophages or myeloid dendritic cells (DCs) [2]. Monocytes are also recruited to sites of infection and mediate anti-microbial activity against viruses, bacteria, fungi and protozoa [3,4]. As first line of defence, both monocytes and macrophages can phagocytose pathogens and foreign particles, release cytokines and generate reactive oxygen species (ROS) in a “respiratory burst” [[5], [6], [7]]. Monocytes and macrophages also contribute to the pathogenesis of inflammatory and degenerative diseases, e.g. atherosclerosis, adipose tissue inflammation and insulin resistance [1,8]. Oxidative stress at the site of inflammation promotes endothelial dysfunction and tissue injury resulting in migration of leukocytes across the endothelial barrier [9]. Since polymorphonuclear neutrophils and macrophages are the main ROS producer in the inflammatory tissue both after acute and chronic infection or tissue damage [[9], [10], [11]], it is reasonable to posit that its amount is tightly regulated. How this occurs is largely unknown.

Previously, we have shown that monocytes compared to macrophages and DCs are hypersensitive to DNA-damaging genotoxins, such as alkylating agents (MNNG and temozolomide), ionizing radiation and oxidizing agents (oxidized low density lipoproteins, tert-butyl hydroperoxide and hydrogen peroxide) [12,13]. The enhanced sensitivity was suggested to be due to a DNA repair defect in monocytes. Thus, the DNA repair pathways for base excision repair (BER) and DNA double-strand break (DSB) repair are down-regulated in human monocytes, which lack the DNA repair proteins XRCC1, ligase IIIα, ligase I, poly(ADP-ribose) polymerase 1 (PARP-1) and the catalytic subunit of DNA-dependent protein kinase (DNA-PK_cs) [12,13]. During the cytokine-mediated maturation of monocytes into macrophages (triggered by granulocyte-macrophage colony stimulating factor; GM-CSF) these DNA repair proteins become up-regulated and, therefore, concomitantly macrophages become repair-proficient and resistant to ROS and other genotoxicants, including ionising radiation [13]. We proposed the hypothesis that the DNA repair defect in monocytes is of biological relevance as it may regulate the amount of monocytes and macrophages in the inflammatory tissue [14].

Downregulation of DNA repair might be a specific property of human monocytes or a general peculiarity of this cell type. Therefore, we set out to determine whether monocytes of mice exhibit the same DNA repair-defective phenotype as human monocytes. To this end, we isolated monocytes from bone marrow of mice and compared them with freshly isolated mouse macrophages. The data revealed that mouse monocytes exhibit downregulation of DNA repair proteins similar to human cells and are killed following temozolomide (TMZ) and total body irradiation (TBR), while macrophages appeared to be resistant. We further show that downregulation occurs during monocyte maturation in precursor bone marrow cells. Overall, the data confirm what we observed in human monocytes, indicating impaired DNA repair is a general property of monocytes.