MGMT deficiency enhances the accumulation of DNA double-strand breaks (DSBs) in macrophages

Because MGMT is responsible for DNA damage repair, loss of MGMT may result in DNA damage in the form of DSBs. γ-H2AX foci formation is a well-known marker of DSBs¹³. Therefore, an immunofluorescence assay for the quantification of γ-H2AX foci formation in BMMs was performed to investigate the effect of MGMT deficiency on the DNA damage response under unstimulated and poly (I:C)-stimulated conditions. The results revealed that the number of γ-H2AX foci per nucleus was significantly greater in the MGMT-KO macrophages than in the WT macrophages under unstimulated conditions (Fig. 2A,B). This result indicated that, compared with WT macrophages, MGMT KO macrophages were more prone to DSBs. In addition, γ-H2AX foci formation increased substantially when WT and MGMT KO macrophages were stimulated with poly (I:C) (Fig. 2B), demonstrating the role of poly (I:C) in exacerbating DNA damage in macrophages and that MGMT is responsible for suppressing DSBs under these conditions.

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Fig. 2

Increased accumulation of DSBs in the MGMT-KO BMMs. (A,B) WT and MGMT KO BMMs were seeded on chamber slides, stimulated with poly (I:C) (20 µg/ml) and incubated further for 24 h. The cells were then fixed and stained for γ-H2AX and nuclear dye. The cells were processed for confocal microscopy analysis of the DNA damage marker γ-H2AX. The data are presented as the means±SEMs from three independent experiments. At least 50 nuclei per condition per independent experiment were quantified. Scale bar 2 µM. ***P<0.001, **P<0.01. (C,D) WT and MGMT KO bone marrow-derived macrophages were treated with poly (I:C) (20 µg/ml) and incubated for 24 h. After incubation, the cells were processed for ATP assays to measure intracellular ATP production or for MTT assays to calculate the cell survival rate. The data are presented as the means±SEMs from at least three independent experiments. **P<0.01 and *P<0.05.

DSBs in nuclei typically lead to the accumulation of DNA damage and ultimately to cell death. Therefore, we measured cell viability using two approaches: an intracellular ATP assay and an MTT assay. The intracellular ATP assay consistently revealed lower luminescence signals in the MGMT-KO macrophages with or without poly (I:C) stimulation (Fig. 2C). In contrast, the MTT assay revealed comparable cell viability between WT and MGMT-KO cells under all conditions, except for a slight reduction only upon poly (I:C) stimulation in the MGMT-KO BMMs (Fig. 2D). Therefore, in the resting stage, the loss of MGMT may result in lower intracellular ATP conditions while preserving cell viability, as measured by mitochondrial reductase activity with the MTT assay.

MGMT localizes to the nucleus and mitochondria in macrophages

We next examined the cellular localization of MGMT puncta with or without poly (I:C) stimulation. Surprisingly, we found that MGMT puncta were present not only in the nucleus but also in the cytoplasm of BMMs under both unstimulated and poly (I:C)-stimulated conditions (Fig. 3A,B). Moreover, the numbers of MGMT puncta in both the nucleus and cytoplasm of macrophages were significantly greater after stimulation with poly (I:C) than in unstimulated conditions (Fig. 3A, B). To pinpoint the cytoplasmic location of MGMT, the mitochondrial marker HSP60 was used for costaining. The colocalization of MGMT and HSP60 increased significantly following poly (I:C) stimulation, indicating that MGMT colocalized with mitochondria (Fig. 3C).

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Fig. 3

Poly (I:C) stimulation increases the MGMT level in the nuclei and mitochondria of BMMs. (A) WT BMMs were stimulated with poly (I:C) (20 µg/ml) and incubated for 24 h. The cells were then fixed and stained for MGMT, HSP60 (a mitochondrial marker) and the nucleus. The cells were processed for confocal microscopy analysis. Scale bar =5 µM. (B) MGMT puncta were quantified in at least 50 cells per condition per independent experiment. The data are presented as the means±SEMs from three independent experiments. *P<0.05. (C) Colocalization of MGMT puncta and Hsp60 quantified in at least 50 cells per condition per independent experiment. The co-localization percentage was calculated using the double positive foci/MGMT+foci. *P<0.05. (D) BMMs from WT mice were stimulated with poly (I:C) as described above, and cytoplasmic and nuclear fractionation was performed. Lysates were subjected to Western blotting to detect MGMT, β-tubulin and histone proteins.

To confirm this unexpected finding, we performed cellular fractionation and Western blotting and observed that MGMT was detectable in both the nuclear and cytoplasmic fractions, with more intense signals after poly (I:C) stimulation (Fig. 3D and Supplementary Fig. S5). Thus, the presence of MGMT puncta in both the cytoplasm (mitochondria) and nucleus indicated that MGMT potentially functions not only in the nucleus but also in the cytoplasm in macrophages. Upon poly (I:C) stimulation, MGMT accumulates in mitochondria, indicating a key function in response to stimulation. It has been previously reported that MGMT is present in the cytoplasm to repair mitochondrial DNA damage caused by alkylating agents in the lymphoblast cell line K-562 ¹⁴, but similar evidence for macrophages has not been presented until now.

MGMT deficiency increases ROS and mtROS production in macrophages

Microbial stimulation of macrophages is linked to changes in mitochondrial metabolism, including changes in the mitochondrial membrane potential and the release of mitochondrial reactive oxygen species (mtROS) and mitochondrial DNA (mtDNA)¹⁵. Furthermore, several studies have reported that excess ROS generated by damaged mitochondria can trigger autophagy^16–18. Therefore, we hypothesized that MGMT KO macrophages may be under mitochondrial stress due to excess ROS production. Using DCFH-DA fluorescence dye to detect ROS, we observed that the fluorescence intensity of the ROS was significantly greater in the MGMT-KO BMMs than in the WT BMMs under all the experimental conditions (Fig. 4A). Poly (I:C) treatment further increased ROS production (Fig. 4A). Moreover, the fluorescence intensity of ROS further increased in BMMs when autophagic flux was inhibited by treatment with bafilomycin A1 (Fig. 4A), indicating a link between autophagy and the clearance of excess ROS.

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Fig. 4

Loss of MGMT increases ROS and mtROS production in BMMs. (A) WT and MGMT-KO BMMs were treated with bafilomycin A1 (500 nM), poly (I:C) (20 µg/ml) or poly (I:C) with bafilomycin A1 and incubated for 24 h. After incubation, the cells were treated with DCFH-DA (10 µM) for 15 min, washed with PBS, and the fluorescence intensity of the ROS was immediately measured at 498/520 nM via a microplate reader. (B,C) Cells were fixed and stained for HSP60 and nuclei and were subsequently stained with MitoSOX reagent (5 µM) for 10 min. The cells were processed for confocal microscopy analysis. Scale bar =2 µM. (C) Corrected total cell fluorescence (CTCF) of MitoSOX was performed on the basis of the images obtained from (B). The data are presented as the means±SEMs from three independent experiments. ***P<0.001, **P<0.01 and *P<0.05.

ROS in macrophages can be generated by damaged mitochondria as well as from other organelle sources¹⁹. Therefore, we used Mitochondrial Superoxide (MitoSOX), which is specific for mitochondria-specific ROS (mtROS), in BMMs with or without bafilomycin A1 treatment to investigate whether the ROS in the MGMT-KO BMMs were derived from mitochondria. The results revealed that the MGMT KO BMMs stained brightly for MitoSOX, which colocalized well with HSP60 (Fig. 4B). The corrected total cell fluorescence (CTCF) of MitoSOX was significantly greater in the MGMT-KO BMMs than in the WT BMMs and was further markedly increased by treatment with bafilomycin A1 (Fig. 4C). Our data thus far strongly indicate that MGMT localizes to mitochondria and regulates ROS production. In the absence of MGMT with poly (I:C) treatment, mtROS increases, a mechanism that is required for autophagy in controlling mtROS levels.

MGMT deficiency enhances autophagy in the BMM

Several studies have reported that ROS, mainly mtROS, can induce autophagy in macrophages and other cell types via the AMPK and mTOR axes^20–23. Hence, we monitored autophagy in the BMM via immunofluorescence staining and Western blotting. In this study, autophagic flux was inhibited by bafilomycin A1 treatment. The increased lipidation of LC3B in the MGMT KO BMMs was supported by the analysis of IF images of LC3B puncta, which revealed that the number of LC3B puncta per cell was markedly greater in the MGMT KO BMMs than in the WT BMMs under all the studied conditions (Fig. 5A,B). The Western blot results also revealed that the band density of the lipidated form of LC3B, called LC3-ii, a marker protein of the autophagosome and autolysosome membrane, was significantly greater in MGMT-KO macrophages than in WT macrophages with or without poly (I:C) treatment when autophagic flux was inhibited with bafilomycin A1 (Fig. 5C and Supplementary Fig. 6). These results strongly indicate that the loss of MGMT in macrophages spontaneously triggers autophagy.

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Fig. 5

Loss of MGMT increases autophagy in BMMs. WT and MGMT-KO BMM were pretreated with bafilomycin A1 (500 nM) for 1 h. The cells were stimulated with poly (I:C) (20 µg/ml) and incubated for 24 h. (A,B) Cells were fixed and stained for LC3B and nuclei. At least 50 cells per condition per independent experiment were quantified for LC3B puncta. The data are presented as the means±SEMs from three independent experiments. Scale bar =5 µM. ***P<0.001, **P<0.01 (C) Cells were treated as indicated, and the cell lysates were processed for Western blotting for LC3Bii.

MGMT deficiency increases mitophagy in macrophages

Clearing damaged mitochondria by autophagy, termed mitophagy, is an essential mechanism for addressing damaged organelles^24,25. However, the link between MGMT and mitophagy has not yet been studied. On the basis of our findings that MGMT deficiency results in increased mtROS and autophagy and the localization of MGMT to mitochondria, we investigated whether mtROS potentially damages mitochondria, which need mitophagy for their clearance, in the MGMT-KO BMMs and in the WT BMMs. To track mitophagy in macrophages, we stained mitochondria, autophagosomes and autophagolysosomes with HSP60, LC3B and LysoTracker™, respectively. We found that under unstimulated conditions, the colocalization of HSP60 with LC3B or LysoTracker™ was significantly greater in the MGMT-KO macrophages than in the WT macrophages (Fig. 6A,B). The percentage of colocalization of HSP-60 with LC3B was further increased in the MGMT-KO cells but not in the WT macrophages after treatment with bafilomycin A1 (Fig. 6A,B), indicating that the MGMT-KO BMMs accumulated more mitochondria containing autophagosomes than did the WT macrophages. Similarly, the percentage of colocalization of HSP60 with LysoTracker™ markedly decreased in both MGMT KO and WT macrophages after treatment with bafilomycin A1 (Fig. 6C,D), indicating the inhibition of autophagolysosome biogenesis due to prevention of the fusion of autophagosomes with lysosomes by bafilomycin A1 ²⁶. Thus, the increased colocalization of mitochondria with autophagosomes and autophagolysosomes in MGMT-KO macrophages and the alteration by an autophagy inhibitor confirmed that MGMT deficiency is actively involved in the induction of mitophagy, potentially to clear damaged mitochondria in macrophages.

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Fig. 6

Loss of MGMT enhances mitophagy in macrophages. (A,B) WT and MGMT KO BMMs were treated with bafilomycin A1 (500 nM) and incubated for 24 h. The cells were fixed and stained for HSP60 and LC3B and processed for confocal microscopy analysis. The colocalization of HSP60 and LC3B was quantified in at least 50 cells per condition per independent experiment. The data are presented as the means±SEMs from three independent experiments. Scale bar =5 µM. ***P<0.001, **P<0.01 and *P<0.05. (C,D) Cells were treated as described above and incubated with LysoTracker Deep Red (100 nM) for 1 h. The cells were then fixed and stained for HSP60 and processed for confocal microscopy analysis. At least 50 cells per condition per independent experiment were quantified for colocalization between lysosomes and HSP60. The data are presented as the means±SEMs from three independent experiments. ***P<0.001, **P<0.01 and *P<0.05.

Mitophagy is a crucial process for mitochondrial homeostasis²⁵. To investigate how excess mitophagy induced by the loss of MGMT affects mitochondrial biomass, mitochondria were stained with MitoTracker™ Green, and the fluorescence intensity was analyzed via flow cytometry and a microplate reader. The mitochondria were also stained with HSP60, and the number of mitochondria per cell was analyzed via confocal microscopy. The results revealed that the fluorescence intensity of MitoTracker™ Green and the number of mitochondria per cell were markedly lower in the MGMT-KO macrophages than in the WT macrophages, which was reversed when mitophagy was inhibited by bafilomycin A1 treatment (Supplementary Fig. S7A–F).

Macrophage stimulation

WT and MGMT KO BMMs were cultured in BMM medium for 24 h at 37 °C and 5% CO₂. The cells were detached with cold PBS, counted, seeded (1×10⁶ cells/well/2000 µl in BMM) in a 6-well plate and incubated for 24 h. The BMM media in each well was replaced with 1000 µl of complete DMEM for nonstimulation or 1000 µl of complete DMEM supplemented with E. coli LPS (100 ng/ml; L2880, Sigma Aldrich, USA) or LPS+IFNγ (100 ng/ml LPS+10 ng/ml IFN-γ) for M1 polarization or with IL-4 (100 ng/ml) for M2 polarization. Recombinant cytokines were obtained from BioLegend (USA). For poly (I:C) stimulation, cells were prepared as described above and treated with poly (I:C) (20 µg/ml; InvivoGen, USA). The macrophages were incubated again for the indicated times. Markers of M1 or M2 macrophage polarization were examined via qRT‒PCR, ELISA and flow cytometry.

qRT‒PCR

WT and MGMT-KO BMMs were treated as indicated, and total RNA was harvested with TRIzol reagent (Invitrogen, USA) and extracted with direct-zol RNA kits (Zymo Research, USA). The quality and concentration of RNA were measured with a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific). At least 200 ng of RNA per sample were converted to cDNA, which was used for quantitative PCR via iQ™ SYBR Green SuperMix (Bio-Rad, USA) according to the manufacturer’s instructions. The relative expression of all target genes (TNFA, IL6, IL1B, IL12p40, IFNB, iNOS, IL10, Fizz1 and ArgI) was normalized to the expression of Actin by the 2^−CT method.

Flow cytometry

WT and MGMT-KO BMMs were treated as described for each experiment. FC receptors were blocked with 10% serum and subsequently washed with FACS buffer before being stained with a mouse anti-mouse CD86 primary antibody or mouse anti-mouse CD40 primary antibody and then with an anti-mouse IgG (H+L) Alexa Fluor 488 secondary antibody (BioLegend), an anti-mouse MHC-II FITC antibody (BioLegend), and an anti-mouse CD206-PE antibody (BioLegend), all in the dark.

For measurement of the mean fluorescence intensity (MFI) of MitoTracker, macrophages in 6-well plates were incubated with MitoTracker Green FM (100 nM) (Thermo Fisher Scientific, USA) for 15 min. For bafilomycin A1+Poly IC treatment, a 1-hr pretreatment with bafilomycin A1 (500 nM) (Sigma Aldrich, USA) was followed by a 24-hr treatment with Poly IC (20 µg/ml) before incubation with MitoTracker. Finally, the stained macrophages were subjected to analysis via flow cytometry. The acquired data were analyzed using FlowJo data analysis software (Tree Star, Inc., USA).

Sandwich ELISA

WT and MGMT-KO BMMs were prepared as described above. The medium in each well was replaced with 1000 µl of complete DMEM only (Unst.) or 1000 µl of complete DMEM with poly (I:C) (20 µg/ml) and incubated again for the indicated times. The culture supernatant was collected for each condition and preserved at -80 °C for use in ELISA. An ELISA MAX™ Standard Set (BioLegend, USA) for TNF-α or mouse IL-6 or IL-10 was used for sandwich ELISA. All reagents used for ELISA, unless otherwise specified, were obtained from BioLegend (USA) and were prepared according to the manufacturer’s instructions.

Cytoplasmic and nuclear protein isolation

To investigate the cellular localization of MGMT, cytoplasmic protein was mildly extracted directly from BMMs on a tissue culture dish using a SimpleChIP^® Enzymatic Cell Lysis Buffer A (Cell Signaling Technology, USA) and incubated on ice until cytoplasmic lysis was observed under the light microscope. The protein lysate was subsequently harvested by centrifugation at 12,000×g to fractionate the cytoplasmic protein and nuclei. The cytoplasmic protein lysate in the supernatant was collected into a new sterile tube, while the remaining pellet (nuclear fraction) was then extracted using high-salt RIPA buffer. The protein lysates were kept at − 80 °C until use.

Western blot

WT and MGMT KO BMMs were prepared, treated with poly (I:C) (20 µg/ml) and incubated for the indicated times. For bafilomycin A1+poly (I:C) treatment, a 1-hr pretreatment with bafilomycin A1 (500 nM) (Sigma‒Aldrich, USA) was followed by a 24-hr treatment with poly (I:C) (20 µg/ml). The cell lysates were collected at the indicated times using RIPA buffer (50 mM Tris HCl pH 7.4, 150 mM (for other proteins) or 500 mM (for histone) NaCl, 5 mM EDTA, 1% nonidet P-40, 0.5% sodium deoxycholate supplemented with protease and phosphate inhibitors (Cell Signaling Technology, USA)). The protein concentrations were measured via a bicinchoninic acid assay using the Pierce BCA Protein Assay Kit (Thermo-Fisher Scientific, USA). Proteins were separated via SDS‒PAGE and subjected to Western blotting as described previously. The antibodies were diluted in PBS with 3% (w/v) skim milk at the following concentrations: rabbit anti-phospho-AMPK-α antibody, 1:1000; rabbit anti-total AMPK-α antibody, 1:1000; rabbit anti-phospho-mTOR antibody, 1:1000; rabbit anti-total mTOR antibody, 1:1000; rabbit anti-LC3B antibody, 1:1000; goat anti-rabbit IgG HRP, 1:4000; and goat anti-rat IgG HRP, 1:4000 (all from Cell Signaling Technology, USA); rat anti-MGMT antibody, 1:1000 (R&D system, USA); mouse anti-actin antibody, 1:10,000 (Merck Millipore, USA); and sheep anti-mouse IgG HRP, 1:4000 (GE Healthcare Life Sciences, USA). The signal was detected via the enhanced chemiluminescence (ECL) method. The relative intensity was analyzed via ImageJ analysis. Uncropped images of the Western blots are shown in Supplementary Fig. S10.

Fluorescence microscopy

WT and MGMT KO BMMs were seeded (3×10⁵ cells/chamber/300 µl in BMM media) on chamber slides (Nunc™ Lab-Tek™ II Chamber Slide™ System; Thermo Fisher Scientific, USA) and incubated for 24 h. BMMs in each chamber were treated as indicated and subjected to staining. To stain acidic vacuoles such as autophagolysosomes in macrophages, the cells were incubated with fluorescence dye (LysoTracker™ Deep Red, Thermo Fisher Scientific, USA) for 1 h before fixation. To stain for mitochondrial superoxide, the macrophages were treated with MitoSOX reagent (5 µM) (Thermo Fisher Scientific, USA) for 10 min before fixation. For fixation, the medium was replaced with 1 ml of fixative (4% paraformaldehyde), and the mixture was incubated for 10 min at room temperature. The fixed cells were permeabilized with 1 mL of 0.1% Triton X-100/PBS for 3 min at room temperature. The cells were subsequently washed 3 times with 1X PBS (5 min each). After washing, the cells were blocked for 1 h at room temperature with blocking solution (3% BSA in 1X PBS). The cells were incubated with a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-LC3B antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-MGMT antibody (Cell Signaling Technology, USA) at a dilution of 1:60, or a mouse anti-HSP60 antibody (DSHB, USA) at a concentration of 1 µg/mL in blocking solution at 4 °C overnight. The cells were washed 3 times (5 min each time) with 1X PBS the following day and incubated with an Alexa 488-conjugated anti-rabbit antibody (Cell Signaling Technology, USA), Alexa 488-conjugated anti-mouse antibody (Thermo Fisher Scientific, USA), Alexa 555-conjugated anti-rabbit antibody (Thermo Fisher Scientific, USA), or Alexa 405-conjugated anti-rabbit antibody (Thermo Fisher Scientific, USA) at 1:500 in blocking solution at room temperature for 2 h. The cells were washed 3 times (5 min each time) with 1× PBS and stained with Hoechst for visualization of the nucleus. After washing with PBS, the slides were mounted with ProLong Gold Antifade Mountant (Life Technologies, USA), and the cells were analyzed via confocal laser scanning microscopy.

Fluorescence intensity measurement

WT and MGMT KO BMMs were cultured and treated as indicated above. For detection of MitoTracker Green fluorescence intensity in macrophages, macrophages in a 96-well black wall/clear bottom plate were incubated with MitoTracker Green (100 nM) for 15 min, followed by fixation with cold methanol and measurement of the fluorescence intensity of MitoTracker Green immediately at 490/516 nM with a multimode microplate reader (Ensight, PerkinElmer, USA). To detect the intensity of the ROS in the macrophages, the cells in the 96-well black wall/clear bottom plate were treated with DCFH-DA (10 µM) (Merck, USA) for 15 min, followed by washing with PBS, and the fluorescence intensity of the ROS was immediately measured at 498/520 nM with a multimode microplate reader.

ATP assay

WT and MGMT KO BMMs treated as indicated were lysed in 50 µL of Mammalian Cell Lysis Solution (MCLS), and the plates were shaken vigorously with an orbital shaker for 10 min. Fifty microliters of substrate solution (ATPLite; PerkinElmer) was added to each well and mixed vigorously (5–7 times) by pipetting. The plates were then incubated in the dark at room temperature for 15 min. After incubation, 50 µL of the sample was transferred from each well of a 96-well culture plate to the respective well of an OptiPlate-96. Finally, the OptiPlate-96 was sealed, and the luminescence was read under standard settings in a multimode microplate reader.

MTT assay

WT and MGMT KO BMMs were treated as indicated. After the incubation period, 10 µl of the MTT reagent (final concentration of 0.5 mg/ml) was added to each well, and the reactions were incubated for an additional 4 h. This step was followed by the addition of 500 µl of DMSO into each well and incubation for 2 h for complete solubilization of the purple formazan crystals. The absorbance of each sample was measured via a microplate reader at 540 nm.

Transcriptomic analysis

All RNA sequencing data were obtained as previously described¹⁰ and can be found under the GEO accession number GSE231419. Subsequently, differential expressed genes (DEGs) in unstimulated WT and MGMT-KO BMM were compared and analyzed using the R package DESeq2 with a p-value cutoff of <0.05. DEGs that were either upregulated or downregulated significantly in MGMT KO macrophages were subjected to Gene Set Enrichment Analysis (GSEA) using GSEA software (GSEA 4.3.2). Gene set mh.all.v2023.1.Mm.symbols.gmt was used as gene set database for GSEA. Gene set was selected as permutation type and number of permutations was 1000. Mouse_Ensembl_Gene_ID_MSigDB.v2023.1.Mm.chip was used as Chip Platform. Significant GSEA pathways were selected with a p-value<0.05 and a false discovery rate (FDR)<0.05.

This research has received funding support from the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (grant number B16F640117) and by Thailand Science Research and Innovation Fund Chulalongkorn University (FF67). AL is supported by the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (grant number B16F640175). This work was partly conducted by the joint research program of the Research Center for GLOBAL and LOCAL Infectious Diseases, Oita University (2023B12). FH and AB are supported by the Second Century Fund (C2F), Chulalongkorn University. This study was partially supported by the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research Innovation (Grant No. B13F670075). PKV is supported by the Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University.