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Macrophages are phagocytic white blood cells, which play an essential role in both innate immunity and adaptive immunity. Uncontrolled macrophages were linked to various diseases such as arthrosclerosis and tumor progression  Atherosclerosis is a chronic inflammatory disease, where macrophages are thought to have malfunction in uptaking and removing cholesterol. In the atherosclerotic lesion, macrophages appear as giant foam cells, cause plaques blocking blood flow and accelerate pro-inflammation [2-3]. In breast cancer setting, tumor associated macrophages (TAMs) should have an anti-tumor properties to benefit the host. Instead, TAMs often promote tumor growth by secreting breast tumor mitogens that direct tumorigenesis and metastasis .
It is well documented that macrophages change phenotypes in response to microenvironment. These phenotypic changes are accompanied with different gene expression profiles which can affect an outcome of an immune response as a whole. Currently, there are at least 3 different phenotypes of effector macrophages which have been reported in mice . The first type is called classical activated macrophages (CA-Mï¦), which arise in response to interferon gamma (IFNï§) together with pathogen associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). They characteristically secreted pro-inflammatory mediators and pro-inflammatory cytokines such as nitrogen and reactive oxygen intermediates with microbicidal activity. Interleukin 12 (IL-12) is a major cytokine produced by this type of macrophages [6-7]. This cytokine is composed of two subunits (p35 and p40) for biological functions  and functions in promoting the development of Th1-type immune response. Because of an effect on promoting Th1 cells, CA macrophages are effective in promoting cell mediated immune responses and provide efficient help for B cell activation and antibody class switching to IgG2a, IgG2b and IgG3 subclasses [9-10].The second population of macrophages have characteristics in tissue repair function and it is called an alternatively activated macrophage (AA-Mï¦) or wound-healing macrophage. This type of macrophages specifically upregulate arginase I (ArgI) expression, which is not found or found at low level in other types of macrophages. IL-4 and/or IL-13, two signature cytokines of Th2 immune response, polarize macrophages to an AA type. Both cytokines promote arginase-dependent formation of L-ornithine and, eventually, fibroblast proliferation and collagen production for tissue repair . When activated macrophages receive an immune complex in the presence of PAMPs, they change phenotype and express high levels of IL-10, IL-12 but lower Arg1. This type of macrophages is called type-2 activated macrophage (Mï¦-ï‰ï‰) or regulatory macrophage. This subset has capacity to present Ag to T cells more efficient than the other two types and seem to induce T cell response toward a Th2 like response, as characterized by Ig class switching to IgG1 and IgE in vivo [5, 12]. The functions of IL-10 secreted by Mï¦-ï‰ï‰ has been implicated in differentiation of T regulatory (Tregs) and is linked to immune suppression such as decreased antigen presentation and major histocompatibility (MHC) class II expression in dendritic cells. It also can cause downregulation of pathogenic Th1, Th2, and Th17 responses .
Notch is a family of evolutionarily conserved transmembrane receptors that regulate various processes which control cell fate decisions, development and cell death. In addition to its well characterized roles in development, these receptors also play roles in effector functions of many immune cells such as in effector phases of T lymphocytes . The signaling is initiated by interaction between Notch receptors and its ligands at the plasma membrane. Activated Notch signaling generates cleaved intracellular Notch which can directly enter the nucleus and bind specifically to DNA binding protein, CSL, and together they regulate the transcription of target genes . In mammals, there are four Notch receptors (Notch1, Notch2, Notch3 and Notch4) and five ligands (Jagged1, Jagged2, Delta-like 1(Dll1), Dll3 and Dll4) . All Notch receptors are proteolytically cleaved and activated by the multi-subunit enzyme called ï§-secretase . The carboxy-terminal notch1 fragment is a further cleaved between Gly1743 and Val1744 [18-19]. The resulting activated cytosolic fragment translocates to the nucleus where it activates transcription. These proteolytical process can be suppressed by ï§-secretase inhibitors (GSI) . Notch receptors and ligands have been shown to be expressed in a different spatio-temporal manner in both inactive and stimulated macrophages . In murine macrophages, GSI treatment suppressed Notch1 upregulation and negatively affected activation of NF-ï«B. While GSI treatment decreased TNFα and nitric oxide production, it increased expressions of MHC class II on cell surface of macrophages upon activation with LPS/IFNï§ . Another studied reported that GSI treatment increases mRNA expression of il10 in M. bovis BCG-infected macrophages . Therefore, it is evident that Notch signaling is involved in regulating effector functions of macrophages.
Because Notch receptors are expressed in macrophages and Notch signaling is reported to be involved in various cell differentiation scenarios, we hypothesize that this signaling may differentially regulate effector functions of macrophages and play a role in their polarization. Understanding the signaling pathway involved in this process not only lead to a new insight in macrophage plasticity but may open a new way to engineer macrophages for better immune responses in the future.
MATERIALS AND METHODS
Bone marrow derived macrophages (BMMï†) from C57BL/6J were used in this study. The procedure for preparing BMMï† has been described elsewhere All procedures involving laboratory animals were conducted according to the guidelines issued by IACUCU of Chulalongkorn University. The animal protocols used in this study have been approved by IACUC of Faculty of Science, Chulalongkorn University (Animal Protocol Rev. No. 0823001).
Cell Culture and Polarizations
BMMï† (3x105 cells) were seeded in 12 well plates and maintained in 1 ml Dulbecco's modified Eagle'smedium supplemented with 10% fetal calf serum (FCS), glutamine, Penicillin-G/Streptomycin To generate 3 different types of macrophage, the protocol described by Edwards et al. was followed (Edwards et al. 2006). Briefly, CA macrophages were generated by priming BMMÐ¤ with rIFNï§ (10 ng/mL) overnight and re-stimulating with LPS (100 ng/ml) for 4 hr for RNA extraction or 6 hr for protein lysate extraction. For AA macrophages, BMMÐ¤ were treated with rIL-4 (100 ng/ml) for duration similar to CA. For Regulatory macrophages, cells were primed with rIFNï§ (100 ng/ml) overnight before being subjected to stimulation with LPS (100 ng/ml) in the presence of and an immune complex (rabbit anti-ovalbumin IgG + ovalbumin (OVA)). To confirm the polarization of macrophages, the expression of il-12 (for CA), il-10 (for regulatory) and argI (for AA) were examined by quantitative realtime RT-PCR.
To inhibit the Notch signaling, GSI (IL-CHO) was used in this study Cells were pretreated with GSI (25 µM) or vehicle control DMSO before subjecting to polarization condition as described above. To confirm that Notch signaling was effectively suppressed by GSI treatment, the expression of Notch1 and cleaved Notch1 were monitored by Western blot. The phenotypes of each type of macrophages were monitored as described above.
Analysis of gene expression by Real-time PCR
Total RNA from macrophages (3x106 cells) was extracted using TRIzol reagent following the manufacturer's instruction (Invitrogen) for reverse transcription, cDNA was generated from 0.1-1.0 µg of total RNA using ReverseAid reverse transcriptase (Fermentas). Realtime-PCR assays were run for 40-45 cycles, Amplification of the housekeeping gene β-actin (Forward 5'-acc aac tgg gac gac atg gag aa-3', Reverse 5'-gtg gtg gtg aag ctg tag cc-3') was used as a quality and loading control. The primers for Notch signaling follows by Narayana and co-worker . To confirm the polarization of macrophages, primers used follows: il10 (Forward 5'-tca aac aaa gga cca gct gga caa cat act gc-3', Reverse 5'-ctg tct agg tcc tgg agt cca gca gac tca a-3'); il12p40 (Forward 5'-aac ctc acc tgt gac acg cc-3', Reverse 5'-caa gtc cat gtt tct ttg cac c-3') and arg1 (Forward 5'-cag aag aat gga aga gtc ag-3', Reverse 5'-caa ata tgc agg gag tca cc-3') [5, 22, 24].
Preparation of cell lysates and Western blot
BMMï† (3x105 cells) were treated as indicated and washed once with ice-cold 1xPBS before rinsing once with 200 µl of buffer A (1 mM EGTA, 1 mM DTT, 50 mM Tris-HCL (pH 7.2), 0.14 M KCl, 2.5mM MgCl2). After addition of 50 µl lysis buffer (0.1ï€¥ Nonidet P-40 in buffer A) and supernatants were transferred to microcentrifuge tubes. Tubes were gently vortexed for 15 s and centrifuged for 5 min at 5,000 rpm, and the supernatants as cell lysate samples were stored at -80°C until use. Depending on the experiment, a loading volume containing from 1.125x105 cells of total cell protein extract was boiled in denaturing buffer and loading dye, and separated on 10% polyacrylamide SDS-PAGE. Proteins were transferred to PVDF membranes (Millipore), which were then processed according to the recommendations provided by the Ab suppliers. Abs was used for Western blot assays were as followed: anti-Notch-1 intracellular region proteins Ab (Santa Cruz Biotech), anti-Cleaved NOTCH1 Ab (Cell Signaling) and anti-Actin Ab (Chemicon) was used for internal protein loading control. There were detected by the ECL method.
Data analysis for statistical significance was done using Student's t-test, except for comparison of expression level between phenotype of macrophages, which was done by multiple comparison of ANOVA (SPSS)
RESULS AND DISCUSSIONS
Polarization of macrophages and expression of Notch 1 in each type
RNA was isolated from the four populations of macrophages at 4 hr after stimulation and analyzed by quantitative RT-PCR (Figure 1A). Expression of arg1, il12p40 and il10 was first examined as they are known markers for each effector macrophages. AA MÐ¤ selectively upregulated arg1 but failed to express il10 and il12p40. In contrast, CA and regulatory MÐ¤ expressed both il10 and il12p40, but CA showed higher level of il12p40 expression and lower level of il10 expression, compared to Regulatory MÐ¤. Conversely, regulatory MÐ¤ expressed highest il10 but almost undetectable il12p40. Unstimulated MÐ¤ expressed all markers at undetectable level. These results confirm the results in previous studied by Edwards and Mosser . For detection of Notch1 and cleaved NOTCH1, protein lysates 6 hr after stimulation were used for analysis by Western blot. As shown in Figure 1B, Notch1 was highly expressed in CA and regulatory MÐ¤, but the level in regulatory macrophages was higher than those in CA MÐ¤. Activation of Notch1 was determined by appearance of cleaved NOTCH1. We found cleaved NOTCH1 in CA MÐ¤ more than in other populations, which correlated with previous studied indicating that Notch1 is involved in pro-inflammatory processes (Figure 1B).
Expression patterns of Notch receptors and ligands in different types of macrophages
Expression Notch2 and Notch3 were found to be high in CA and regulatory macrophages, respectively (Figure 2). Expressions of Notch4 were below detectable level in all subsets of macrophages (data not show). After polarization of macrophages, the ligands expressions were determined by quantitative RT-PCR. We examined all five Notch ligands, i.e. dll1, dll3, dll4, jagged1 and jagged2 (Figure 2 B-C). For comparison of relative expression levels, unstimulated macrophages were used as base line. The expression levels could be summarized as follows: dll1: Reg > CA > AA, dll4: AA > Reg > CA, jagged1: CA > Reg > AA and jagged2: AA > CA > Reg. But the expression of dll3 was not detectable (data not show). Summary of expression of Notch receptors and ligands was shown in Figure 2C. Expression of dll1 and jagged1 were found to be highest in CA and Regulatory, consistent with previous studies in murine and human monocyte bone-marrow-derived macrophages which found that Jagged1 and Dll1 were induced by TLR4 ligation and regulated by IFNï§ . Expression of dll4 was found to be highest in AA and regulatory macrophages, consistent with previous report suggesting that Dll4 induces Notch activation, resulting in increased expression of pro-inflammatory genes . Moreover, we found that expression of jagged2 was significantly increased in AA macrophages.
Figure 1 Polarization of macrophages and Notch1 expression in each type
(A) Relative mRNA expression by Real-time PCR. Arginase1 (Arg1), Il12p40 and Il10 were used to confirm phenotype of AA, CA and regulatory macrophages, respectively.
(B) Western blot of Notch1 and cleaved Notch1 in each type of macrophage.
Figure 2 Expression profiles of Notch receptors and ligands in each type of activated macrophages
(A) Expression level of Notch2 and Notch3 and (B) Notch ligand in different type of activated macrophages as determined by quantitative RT-PCR. (C) Summary of relative mRNA expression of Notch receptors and ligands in each type of activated macrophages. (*) indicate statistical significance if p < 0.05.
Figure 3 Effect of GSI treatments on polarization of macrophages
(A) Macrophages were pretreated with IL-CHO or vehicle control DMSO as described in materials and methods before subjecting to polarization. Protein lysates were analyzed for Notch1 (left) and cleaved Notch1 (right) by Western blot. (B) Quantitative RT-PCR of marker genes in each type of macrophages was examined after cells were treated with IL-CHO or DMSO; Unstimulated macrophages (a), CA macrophages (b), AA macrophages (c) and Regulatory Macrophages (d). The statistical analysis was carried out using parametric 1-tail of Student's t-test for compared effect of treatments, (*) indicated where statistical significance if p < 0.05.
Inhibition of Notch signaling affects expression of makers for each type
GSI inhibits the activity of gamma-secretase and suppresses Notch receptor processing. After treatment with GSI, decreased in both Notch1 and cleaved Notch1 were observed (Figure 3A). Unexpectedly, in regulatory MÐ¤, GSI treatment resulted in more uptake of an immune complex which interfered with the Western blot to detect cleaved Notch1. When mRNA expression profiles were examined, it was found that GSI treatment has differential effects on marker gene expression (Figure 3B). GSI treatment caused decreased il10 and il12p40 mRNA expression in CA and regulatory macrophages, respectively. Moreover, GSI treatment did not affect arg1 mRNA level in AA macrophages. Previous reported by Wang et al., show that GSI treatment of macrophages affected level of IL-12 expression in M1 or classically activated macrophages which are consistent with these results. Therefore, we concluded that Notch signaling may function in controlling of effector macrophages differentiation.
This study demonstrated that Notch signaling is involved in differentiation of effector macrophages, especially in CA and regulatory macrophages. Differential expression of Notch receptors and ligands were found in 3 different types of macrophages. GSI treatment strongly affected marker gene expression of each type of macrophages. How Notch signaling controls expression of il12 and il10 expression is currently not known. Notch signaling may function to affect gene expression directly or indirectly by regulating other signaling pathways such as MAPK pathway. Understanding the interaction among these signaling pathways may open a new way to engineer macrophages for treatment and better immune responses in the future.
This research was supported by research grant from Thailand Research Fund (TRF) and Chulalongkorn University Rachadapisek Somphot Endowment Fund.