Unrepaired DNA damage in macrophages causes elevation of particulate matter- induced airway inflammatory response

In vivo or in vitro exposure to PM induces DNA breaks in macrophages

Accumulating evidence suggests that various levels of PM exposure induce significant DNA damage in occupational populations and in cultured cell lines [27-29]. To further examine the genotoxic effects of PM on the airway epithelium or macrophages in vivo, male C57BL/6 mice, aged 6-8 weeks, were instilled intratracheally with PM at 100 μg·d^-1·mouse^-1 for 2 days. As revealed by TUNEL assay, the short-term PM exposure triggered remarkable DNA damage following apoptosis in the lung tissues (Fig. 1A). Surprisingly, DNA damage tended to be distributed in the peripheral pulmonary zone while the airway epithelium displayed inappreciable damage effects. Given that the TUNEL assay is limited in its sensitivity and specificity in detecting DNA damage following apoptosis [30], we used other assays in the following steps to further confirm the effects.

We next sought to confirm the locations of damage foci by marking alveolar macrophages (AMs) with CD68 staining (Fig. 1B). Double immunofluorescence labelling partially localized damage foci to AMs. These findings indicated that macrophages played important roles in PM-induced genotoxicity, at least during acute exposure. Consistent with in vivo findings, PM induced significant DNA damage in BMDMs, as revealed by the DNA percentage in the comet tail in a dose–dependent manner (Fig. 1C). In summary, these results clearly demonstrate that PM could induce DNA damage in macrophages both in vivo and in vitro.

PM exposure induces DDR signaling in macrophages, involving upregulation of the RAD50 sensor protein

We next examined DDR signaling in the lung tissues and BMDMs. Similar to that seen where the DNA damage occurred, phosphorylated histone H2AX(γH2AX) and RPA foci, DDR markers, primarily occurred in peripheral lung tissue rather than the airways. Quantification of γH2AX and RPA also demonstrated a marked elevation of DDR in response to PM exposure. (Fig. 2A and B). Likewise, quantification of γH2AX-positive cells (≥5 foci per nucleus) revealed higher levels of DNA damage in BMDMs upon PM stimulation (Fig. 2C). PM exposure also slightly increased the frequency of 53BP1-positive cells (≥5 foci per nucleus) in BMDMs (Fig. 2D). Because these events require rapid recognition of the DDR sensor, we next examined the expression patterns of RAD50, a subunit of the MRN complex, using immunofluorescence and western blots. As shown in (Fig. 3A and B), RAD50 protein levels increased with PM exposure in a dose-dependent manner. Because phosphorylation of H2AX requires activation of the phosphatidylinositol-3-OH-kinase-like entities ATM and ATR kinases, we next determined whether the observed increase in γH2AX was related to the autophosphorylation of ATM. As shown in (Fig. 3C), immunofluorescence staining demonstrated an increase in phosphorylated ATM protein at serine 1981 in BMDMs upon PM exposure.

Genetic deletion of RAD50 in macrophages increases cytokine production following PM stimulation

Our findings suggest that the DDR sensor RAD50 could have important functionalities. According to previous studies [18,31,32], we hypothesized that the absence of a DDR sensor in mice could lead to persistent, unrepaired DNA damage, and thus augments fine PM-induced inflammatory cytokine secretion. To corroborate hypothesis, we used BMDMs from mice with RAD50 deletion in myeloid cells (RAD50^flox/flox-LysM^cre) and wildtype (WT) mice (RAD50^flox/flox). To test this idea, we first checked direct DNA damage using the alkaline comet assay. The BMDMs from RAD50^flox/flox-LysM^cre mice displayed relatively high basal levels of DNA damage, which was further increased after PM exposure, contrasting with results found with WT controls (Fig. 4A and B). We next detected the inflammatory response. Treatment with PM induced significantly higher levels of Cxcl1, Cxcl2 and Ifn-γ in RAD50^flox/flox-LysM^Cre macrophages compared with those in WT cells, as determined using real-time PCR (Fig. 4C). In summary, loss of DDR sensor RAD50 in macrophages augments DNA breaks, resulting in amplified inflammatory cytokine response in vitro.

Chemical inhibition of Mre11, a central MRN complext component, promotes PM-induced cytokine production

To further confirm the role of the DDR sensor in the inflammatory response in macrophages, we used mirin, a potent MRN complex inhibitor [33], to block the MRN complex by inhibiting Mre11-associated exonuclease activity. Compared with the control group, BMDMs exposed to PM after treatment with mirin (administered dose of 100 μM) produced marked elevations in both mRNA and cytokine protein levels, including Cxcl1, Cxcl2 and Ifn-γ (Fig.5A and B). These results strengthen the idea that the disruption of MRN complex causes elevated inflammatory response in BMDMs upon PM exposure.

RAD50^fl/fl-LysM^Cre mice display exacerbated airway inflammation in response to PM exposure

To further elucidate the role of the DDR sensor in airway inflammation in vivo, we challenged RAD50^flox/flox -LysM^cre mice and their WT littermates with PM or normal saline(NS) via intratracheal instillation. The total number of inflammatory cells and neutrophils in the bronchoalveolar lavage fluid from RAD50^flox/flox-LysM^cre mice was significantly higher relative to the littermate controls (Fig. 6A). Analysis of lung extracts revealed increased mRNA and protein levels of cytokines such as Cxcl1, Il6, Il17 and Ifn-γ in RAD50^flox/flox-LysM^cre mice following treatment with PM (Fig. 6B). Furthermore, PM-induced airway inflammation was significantly upregulated in RAD50^flox/flox-LysM^cre mice, and this was confirmed using immunohistological analysis (Fig. 6C and D). These results demonstrate that RAD50^flox/flox-LysM^cre mice with impaired DNA damage sensor display exacerbated proinflammatory responses.

Mice

Mice were maintained on a mixed C57BL/6 and 129Sv background. Myeloid cell-specific RAD50 conditional knockdown mice (RAD50^flox/flox-LysM^cre) were obtained by crossing RAD50^flox/flox mice with mice expressing Cre recombinase under the control of the Lysozyme promoter (LysM^cre). LysM^cre-negative, RAD50^flox/flox littermates served as controls. The RAD50^flox/flox mice were obtained from Jackson Laboratory, and the LysM^cre mice on the C57BL/6 background were a generous gift from Dr. Gen-Sheng Fen (University of California at San Diego, CA, USA). The WT C57BL/6 mice were purchased from the Laboratory Animal Center of Zhejiang University (Hangzhou, China). Four- to five-week-old mice were typically used for in vitro experiments. Six- to eight-week-old mice were used for in vivo studies. All mice were maintained in a specific-pathogen-free facility. All experimental protocols were approved by the Ethical Committee for Animal Studies at Zhejiang University.

Cell preparation

Bone marrow was harvested and cultured for 6 days in complete Dulbecco's Modified Eagle Medium containing 10% heat-inactivated FBS (Gibco 10099-141), 1% penicillin-streptomycin (Gibco, 15140-122) and 10 ng·mL^-1 mouse M-CSF (R&D Systems, 416-ML-010). On day 6, a homogeneous population of adherent macrophages was obtained after 6 days of culture (>95% CD11b and F4/80). In a subset of experiments, BMDMs were treated with PMs at indicated concentration and time points. In experiments in which MRN complex activity was blocked chemically, mirin (100 nM; Selleck) was used.

In vitro and in vivo PM exposure

Ambient urban PM, Standard Reference Material 1649b, was obtained from the National Institutes of Technology (Gaithersburg, MD, USA). The PM was suspended and sonicated in sterile saline at a concentration of 2 mg·mL^-1 (mass/volume). In vitro, BMDMs were treated with PMs at 100 μg·mL^-1. In vivo, mice were instilled intratracheally with PM 100 μg (in 50 μL saline) per day for 2 days.

RNA isolation and quantitative real-time PCR analysis

From cells and lung tissues, RNA was isolated using Trizol (Takara Biotechnology). Reverse transcription was performed using reverse transcription reagents (Takara Biotechnology). The expression of mouse Cxcl1, Cxcl2 and Ifn-γ were measured using quantitative real-time PCR with SYBR Green Master Mix (Takara Biotechnology) on a StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA). All protocols were performed according to manufacturer’s instructions. Primer sequences used are as follows:

Actin B:

Forward: 5′-GTCCACCGTGTATGCCTTCT-3′,

Reverse: 5′-CTCCTGGTGTCCGAACTGAT-3′;

Cxcl1:

Forward: 5’-CTGGGATTCACCTCAAGAACATC-3’,

Reverse r: 5’-CAGGGTCAAGGCAAGCCTC-3’;

Cxcl2:

Forward: 5’-TGTCCCTCAACGGAAGAACC-3’,

Reverse: 5’-CTCAGACAGCGAGGCACATC-3’;

Il6:

Forward: 5’-CTGCAAGAGACTTCCATCCAG-3’,

Reverse: 5’-AGTGGTATAGACAGGTCTGTTGG-3’;

Ifnγ:

Forward: 5’-ACAGCAAGGCGAAAAAGGATG-3’,

Reverse: 5’-TGGTGGACCACTCGGATGA-3’;

Il17:

Forward: 5’-TCAGCGTGTCCAAACACTGAG-3’,

Reverse: 5’-CGCCAAGGGAGTTAAAGACTT-3’;

Chemicals and reagents

Antibodies against CC10 (Santa Cruz Biotechnology, sc-9772), CD68(Servicebio, GB11067), Actin B (Santa Cruz Biotechnology, sc-47778), RAD50(Abcam, ab89), γH2AX (EMD Millipore, 05-636), RPA (Abcam, ab2175), pATM (EMD Millipore) and 53BP1 (EMD Millipore, MAB3802) were used. Antibodies for immunofluorescence were diluted at 1:1000 in 5% BSA (Sigma-Aldrich, B2064) except for the pATM which was diluted at 1:500. Antibodies for western blot were diluted at 1:1000 in 5% nonfat dried milk. All primers used in the study were synthesized by Sangon Biotech,Shanghai. ELISA kits for mouse Cxcl1 (MKC00B), mouse Cxcl2 (MM200), mouse Il6 (M6000B), mouse Il17(R&D, M1700) and mouse Ifn-γ (R&D, MIF00) were purchased from R&D systems. MRE11 inhibitor mirin was purchased from Selleck (S8096), and its final concentration in culture medium was 100μM.

ELISA

The concentrations of Cxcl1, Cxcl2, Il6, Il17and Ifn-γ in cultured medium and bronchoalveolar lavage fluid supernatants were determined using ELISA kits from R&D Systems following manufacturer’s instructions.

Immunofluorescence

Bone marrow–derived macrophages were seeded on cleaned and autoclaved glass coverslips. Following the indicated treatment, the cells were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde solution for 30 min at room temperature. Subsequently, the cells were permeabilized with 0.1% Triton-X-100 (Sigma-Aldrich, LC262801) in PBS for 10 min at 4°C and blocked with 5% BSA (Sigma-Aldrich, B2064) for 30 min at room temperature followed by washing with PBS. Then the cells were incubated with primary antibody overnight at 4°C. After washing the cells with PBS, Alexa Fluor®555-conjugated goat anti-mouse IgG (HCL) antibody (Invitrogen, A28180) solution was added and incubated for 1 h at room temperature, washed with PBS. All DNA were counterstained with DAPI. Fluorescent images were captured with a Zeiss LSM laser scanning confocal microscope. For each condition, 10 fields of at least 14 cells each were quantified, and cells with 5 or more foci were counted as positive.

For the double immunofluorescence labeling, cells were fixed, permeabilized and blocked as previously described. Then incubate cells in the mixture of two primary antibodies (e.g.goat against CC10 and mouse against γH2AX or RPA) in 5% BSA in PBST in a humidified chamber overnight at 4°C. Wash the cells three times in PBS, 5 mins each wash. Incubate cells with the mixture of two secondary antibodies which are raised in different species (with two different fluorochromes, e.g. Alexa Fluor®488-conjugated goat anti-mouse IgG (HCL) antibody and Cy3-conjugated donkey Anti-goat IgG (H+L)) in 5% BSA for 1 hr at room temperature in dark. DNA was counterstained with DAPI. Finally the samples were analyzed under fluorescence microscope as described above.

Comet assay

Bone marrow–derived macrophages were cultured as described. After incubation, cells were harvested, processed and analyzed using a Comet Assay Kit (Trevigen) according to manufacturer's protocol. After assay, slides were stained with DAPI and viewed by epifluorescence microscopy. Cell images were analyzed using CASP software (downloaded from www.casplab.com). Among the comet parameters, we report the percent DNA in the tail (tail DNA%) as a marker of DNA damage. Two hundred comets were scored randomly for each indicated treatment. All experiments were performed in triplicate.

TUNEL assay

The TUNEL (terminal deoxynucleotidyl transferasemediated dUTP nick end labeling) assay was performed to detect DNA fragmentation in bone marrow–derived macrophages by using a commercial kit (Roche Diagnostics Limited, In situ Cell Death Detection kit, fluorescein, 11684795910). Paraffin-embedded lung tissue sections were permeabilized in 0.1% Triton X-100(Sigma-Aldrich, LC262801), blocked nonspecific binding with 5% bovine serum albumin (BSA) in PBS for 1h at room and then incubated with TUNEL reaction mixture containing label solution and enzyme solution in a humidified atmosphere for 60min at 37°C. Nuclei were counterstained with DAPI. Samples were analyzed under fluorescence microscope for DNA breaks. The TUNEL positive rate was determined according to the formula: (number of cell with green staining/total number cells with DAPI staining) × 100%.

To perform a TUNEL assay with CD68 co-staining, Lung tissues were permeabilized and blocked as described previously. Incubate coverslips in a humidified chamber with CD68 (dilution at 1:1000 in 5% BSA) overnight at 4°C. incubated with 50μL TUNEL reaction mixture in a humidified atmosphere for 60min at 37°C following wash with PBS. and then 50μL of 555-conjugated goat anti-rabbit IgG (HCL) for 1h at 37°C. Rinse coverslips 3× 10min in PBS. DNA was counterstained with DAPI. Analyze coverslips under fluorescence microscope as described above.

Western blot

Whole-cell extracts were prepared by lysis and sonication of cells in RIPA buffer (Beyotime, P0013B) containing phosphatase inhibitors (Roche Diagnostics GmbH, 04-906-837-001) and protease (Roche Diagnostics GmbH, 04-693-116-001). Lysates were run on SDS–PAGE, then transferred onto PVDFmembrane (millipore, IPVH00010), imunoblotted with relevant antibodies using standard methods. Actin B served as a protein loading control.

Histological analysis

Following each indicated treatment, lung tissues were fixed and stained with hematoxylin and eosin to demonstrate general morphology. Slides were digitized using an Olympus BX53 inverted microscope (Olympus, Melville, NY). The severity of inflammation was graded semiquantitatively according to published guidelines [46] as follows: 0, normal; 1, few cells; 2, a ring of inflammatory cells 1 cell layer deep; 3, a ring of inflammatory cells 2–4 cells deep; 4, a ring of inflammatory cells of >4 cells deep.

Statistical analysis

Statistical significance was calculated using the two-tailed Student t test unless otherwise indicated. Prism 6 software was used to generate graphs and statistical analyses. Statistical significance was recognized when p ≤ 0.05.