A simple method to increase the proportion of bone marrow-derived macrophages positive for M-CSFR using the reducing agent dithiothreitol (DTT)

Graphical abstract

Treatment with a reducing agent dithiothreitol (DTT, 1 mM) for 24 h increased the proportion of BM-MF that were positive for M-CSFR to 51.7 AE 7.3% ( Fig. 2A). This increase following DTT treatment was time dependent ( DTT is known to be both a reducing reagent and an endoplasmic reticulum (ER) stress inducer. Treatment with another reducing agent, 2-mercaptoethanol (1 mM), for 24 h did not alter the proportion of BM-MF that were positive for M-CSFR (Fig. 3, vehicle control; 1.0 AE 0.0, 24 h of 2mercaptoethanol; 0.99 AE 0.07-fold). Treatment for 24 h with a different ER stress inducer, tunicamycin (0.5 mg/mL), led to a decrease in the proportion of BM-MF that were positive for M-CSFR (Fig. 4, vehicle control; 1.0 AE 0.1, 24 h of tunicamycin; 0.17 AE 0.06-fold). These results suggest that DTT, but not a reducing reagent, or an ER stress inducer increases the proportion of BM-MF that are positive for M-CSFR.
DTT-treated BM-MF can lead to the downregulation of M-CSFR. It has been previously reported that lipopolysaccharide (LPS) downregulates M-CSFR [7,8]. We have also observed, as shown in Fig. 5A, that treatment with 2 mg/mL of LPS for 24 h decreased the

Additional information
The study conformed to the guidelines given in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The experimental protocol was approved by the Animal Care and Use Committee of Juntendo University.
Background: Macrophages play a role in the pathogenesis of atherosclerosis [9,10]. M-CSF (CSF-1) regulates monocyte/macrophage survival, proliferation, differentiation, and migration via the activation of its receptor (M-CSFR) [11][12][13]. Studies have demonstrated a decrease in aortic atherosclerosis in both M-CSF-/-/ApoE-/- [14] and M-CSF-/-/LDLR-/- [15] mice. It has also been reported that an M-CSFR-neutralizing antibody resulted in both a decrease in atherosclerosis [16] and pharmacologic inhibition of M-CSF signaling via a specific inhibitor of M-CSFR (GW2580), which also resulted in a decrease in atherosclerosis [17,18]. In this manner, the contributions of M-CSF and M-CSFR to atherogenesis, as well as the underlying mechanisms that are involved, have been revealed. It has been reported that treatment with M-CSF, phorbol ester (12-O-tetradecanoylphorbol-13acetate (TPA)), and LPS downregulates M-CSFR expression in macrophages [19][20][21][22][23][24]; however, few studies of the relationship between the downregulation of M-CSFR and atherosclerosis have been reported. One of the contributing factors is the small proportion of macrophages that is positive for M-CSFR on the plasma membrane [1]. Thus, there is a need for a method to increase the number of macrophages that are positive for M-CSFR and investigate the mechanisms underlying the downregulation of M-CSFR during atherogenesis. In the present study, we have shown that treatment of BM-MF with DTT increased the proportion that was positive for M-CSFR and that the upregulation of M-CSFR on BM-MF can be abrogated by treatment with LPS. Here, we propose a simple method to increase the number of M-CSFR-positive BM-MF that utilizes the reducing agent DTT that may be useful in the investigation of the relationship between the downregulation of M-CSFR and some diseases such as atherosclerosis.
Future perspectives to reveal the relationship between the downregulation of M-CSFR and various diseases: We propose that DTT could be successfully utilized to investigate the relationship between the downregulation of M-CSFR and atherosclerosis. However, when using DTT in animal models in vivo, any possible side effects of DTT should be considered. For example, we may need to consider any cytoprotective effects that DTT may exert via an increase in the production of hydrogen sulfide (H 2 S) because there is now an abundance of scientific evidence that suggests that, despite its reputation as a noxious gas with wide-ranging cytotoxic effects, H 2 S, in fact, has cytoprotective effects [25].
H 2 S also protects neurons from oxidative stress by restoring the levels of glutathione, a major intracellular antioxidant, via enhancement of the activity of g-glutamylcysteine synthetase and the  transport of cysteine and cystine [26,27]. Additionally, it protects cardiomyocytes from ischemia/ reperfusion injury by contributing to the preservation of mitochondrial function [28]. The production of H 2 S in mammalian systems has been attributed to three key enzymes: cystathionine β-synthase (CBS), cystathionine g-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) [29][30][31]. 3MST produces H 2 S from 3-mercaptopyruvate (3 M P), which is generated by cysteine aminotransferase (CAT) from cysteine and α-oxoglutarate (α-KG) [31][32][33][34]. 3 M P provides sulfur to the active-site cysteine residue of 3MST to produce persulfide, which, in turn, releases H 2 S in the presence of DTT [31,35]. Thus, the use of DTT, with due consideration to its possible cytoprotective effects, may facilitate the investigation of the relationship between the downregulation of M-CSFR and various diseases, including atherosclerosis, during future studies. Discrepancy of the M-CSFR expression between this report and previous report: It was reported that 65.4 AE 3.0% of BM-cultured MF express M-CSFR [1], and we showed that 24.4 AE 4.6% of BMcultured MF express M-CSFR. It was also reported that 3.6 AE 0.2% of peritoneal cultured macrophages express M-CSFR [1], and we showed that 95.3 AE 0.8% of isolated peritoneal macrophages express M-CSFR in this report. M-CSF, phorbol ester, and LPS are known to downregulate M-CSFR expression in macrophages [19][20][21][22][23][24]. Thus, differences in the culture condition (especially serum differences) might cause the difference in M-CSFR expression in BM-cultured MF. The discrepancy in peritoneal macrophages is consistent with our data that the proportion of macrophages positive for M-CSFR is higher in isolated cells (37.8 AE 2.7% of isolated BM-MF; Fig. 1) than in cultured cells (24.4 AE 4.6% of BM-cultured MF; Fig. 2). Thus, the discrepancy in peritoneal macrophages might be explained by the difference between cultured cells and isolated cells.