Small-Molecule Inhibitor of 8-Oxoguanine DNA Glycosylase 1 Regulates Inflammatory Responses during Pseudomonas aeruginosa Infection

Shugang Qin, Ping Lin, Qun Wu, Qinqin Pu, Chuanmin Zhou, Biao Wang, Pan Gao, Zhihan Wang, Ashley Gao, Madison Overby, Jinliang Yang, Jianxin Jiang, David L. Wilson, Yu-ki Tahara, Eric T. Kool, Zhenwei Xia and Min Wu
J Immunol October 15, 2020, 205 (8) 2231-2242; DOI: https://doi.org/10.4049/jimmunol.1901533

Key Points

  • OGG1 inhibitor SU0268 facilitates antibacterial immunity in PA14 infection.

  • SU0268 mitigates bacterial infection via an mtDNA-mediated cGAS pathway.

  • SU0268 attenuates lung injury caused by PA14 infection in vivo.

Abstract

The DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which excises 8-oxo-7,8-dihydroguanine lesions induced in DNA by reactive oxygen species, has been linked to the pathogenesis of lung diseases associated with bacterial infections. A recently developed small molecule, SU0268, has demonstrated selective inhibition of OGG1 activity; however, its role in attenuating inflammatory responses has not been tested. In this study, we report that SU0268 has a favorable effect on bacterial infection both in mouse alveolar macrophages (MH-S cells) and in C57BL/6 wild-type mice by suppressing inflammatory responses, particularly promoting type I IFN responses. SU0268 inhibited proinflammatory responses during Pseudomonas aeruginosa (PA14) infection, which is mediated by the KRAS–ERK1–NF-κB signaling pathway. Furthermore, SU0268 induces the release of type I IFN by the mitochondrial DNA–cGAS–STING–IRF3–IFN-β axis, which decreases bacterial loads and halts disease progression. Collectively, our results demonstrate that the small-molecule inhibitor of OGG1 (SU0268) can attenuate excessive inflammation and improve mouse survival rates during PA14 infection. This strong anti-inflammatory feature may render the inhibitor as an alternative treatment for controlling severe inflammatory responses to bacterial infection.

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen that causes many serious infectious diseases (1). Lung fibrosis and chronic lung injury, which can extensively induce other lung diseases and accelerate disease progression, are two abundant outcomes secondary to P. aeruginosa infection, which is difficult to treat and eradicate invading pathogens (2). Numerous studies have shown that P. aeruginosa infection is positively associated with excessive activation of inflammation (3). Therefore, regulation of the inflammatory response is crucial for the treatment of P. aeruginosa infections and associated lung diseases (4).

Reactive oxygen species (ROS) that are generated during bacterial infection induce genome damage by the formation of oxidatively damaged DNA bases, the most frequent of which is 8-oxo-7,8-dihydroguanine (8-oxoG) (5, 6). To ameliorate harmful mutagenic effects of this damage, the base 8-oxoG is excised from genomic and mitochondrial DNA (mtDNA) by 8-oxoguanine DNA glycosylase-1 (OGG1) through the DNA base excision repair (BER) pathway (7). OGG1 is a DNA repair enzyme that is closely linked to oxidative stress and inflammation (8). Mice engineered to lack the OGG1 enzyme develop normally (9). Although they accumulate higher levels of 8-oxoG in their DNA, they are otherwise relatively healthy and exhibit normal lifespans (10). Importantly, in tests of inflammation, Ogg1−/− mice were shown in an early study to blunt proinflammatory responses to LPS and allergens (11). The mice showed a lowered extent of neutrophil infiltration, decreased levels of Th1 cytokines IL-12 and TNF-α, and elevated levels of protective Th2 cytokines IL-4 and IL-10 (12).

Multiple connections between the function of OGG1 and inflammation have been documented. DNA promoter region binding by OGG1 increased the expression levels of chemokines/cytokines, including Cxcl-2 and TNF-α (8, 13). Thus, the prevention of DNA binding by a small-molecule OGG1 inhibitor may be expected to inhibit this proinflammatory signaling. Efforts to develop OGG1 inhibitors have been reported recently, including compound O8, which inhibits DNA lyase activities of OGG1 but does not inhibit DNA binding (14) and SU0268, which inhibits DNA binding and base excision by the enzyme (15). Recently, Visnes et al. (16) reported the development of a third small-molecule inhibitor of OGG1, TH5487, and tested it in a mouse model of inflammation induced by challenges with LPS or TNF-α. The study documented that TH5487 decreased levels of proinflammatory cytokines and chemokines, including IL-6, Tnf, and Cxcl2, and did so in a dose-dependent manner. It also decreased neutrophil infiltration in blood and lung fluid and increased mouse survival rates after LPS exposure. The study provided further validation of OGG1 as a potential target for inflammation (16). To date, no studies of the effects of OGG1 inhibitors on inflammation induced by bacterial infections have been reported.

Inflammation resulting from pathogen infections is documented to be mediated, at least in part, by the intracellular release of mitochondrial and microbial DNA (17). The cyclic GMP–AMP synthase (cGAS), which is dependent on the binding of mtDNA arising intracellularly and extracellularly, is closely related to immune function during bacterial and viral infections (18). Activated cGAS triggers the immune response through regulation of the production of type I IFNs in generating the second messenger 2′3′-cyclic-GMP–AMP (cGAMP) (19). It is well known that OGG1 is involved in the process of DNA damage repair during bacterial infection. Studies have demonstrated that microbial DNA released into the cytoplasm is the major agonist of cGAS (20). However, the interactions between OGG1-mediated pathways and cGAS are currently unclear. In addition, whether OGG1 can be a potential candidate target for anti-inflammatory treatment during bacterial infection remains untested.

The compound SU0268, the most potent existing OGG1 inhibitor, is a promising small-molecule candidate to probe the significance of OGG1 inhibition in downregulating clinically harmful inflammatory responses. The cell-permeable compound exhibits an in vitro IC50 of 59 nM, is selective for OGG1 over other BER enzymes, and shows low toxicity at concentrations 10 μM and below. The compound has been documented to inhibit DNA binding by OGG1, unlike previous OGG1 inhibitors. However, the activity of SU0268 in potentially influencing inflammatory responses has not yet been explored (15).

In the current study, we investigated the potential role of the compound SU0268 in modulating inflammatory response and bacterial clearance both in vitro and in vivo after P. aeruginosa reference strain 14 (PA14) infection. Our study demonstrates that the compound inhibits murine OGG1 activity in a mouse cell line. We found that SU0268 can potently inhibit inflammatory responses and mitigate bacterial infection. Our data show new, to our knowledge, evidence of the relevance of OGG1 as a target in inflammation and the compound SU0268 as a useful tool in preclinical models of this immune response.

Materials and Methods

Mice

C57BL/6N mice (6–8 wk) were purchased from the Harlan Laboratory. The mice were maintained in the animal facility at the University of North Dakota for at least 2 wk before starting the experiment. Both male and female were randomly selected (age of mice ranged from 6 to 8 wk). All animal studies were approved by the University of North Dakota Institutional Animal Care and Use Committee and performed in accordance with the animal care and institutional guidelines (21).

Primary cells and cell lines

Mouse primary alveolar macrophage cells, bone marrow–derived macrophage (BMDM) cells and bronchoalveolar lavage fluid (BALF) were obtained through alveolar lavage as previously described (22), cultured in RPMI 1640 (Life Technologies) supplemented with 10% FBS (Life Technologies), and incubated on a culture plate for 4 h at 37°C/5% CO2 incubator to allow attachment of the macrophages. Nonadherent cells were removed by washing with sterile PBS. BMDM cells were induced into mature macrophages by adding recombinant mouse GM-CSF (PMC2011; Thermo Fisher Scientific, Waltham, MA) in the medium. Alveolar lavage fluid murine (MH-S) cells and J744.1A cells were obtained from American Type Culture Collection (Manassas, VA) and cultured following the manufacturer’s instructions.

Bacteria and bacterial infection assay

The wild-type PA14 was a gift from S. Lory (Department of Molecular Genetics, Harvard Medical School). Bacteria were grown to the midlogarithmic phase (OD600 = 0.5–0.6) in lysogeny broth at 37°C with 220 rpm shaking. The number of bacteria was determined by measuring the OD value (1 OD = 1 × 109 cells/ml). Mammalian cells were infected PA14 in a multiplicity of infection of 20:1 bacteria/cell ratio. C57BL/6N mice were anesthetized with ketamine (45 mg/kg) and intranasally infected with 6 × 106 CFU of PA14.

SU0268 treatment

SU0268, synthesized as previously described (15), was diluted in pure ethanol, then MH-S cells were treated with SU0268 for varied times before PA14 infection for 2 h (pure ethanol as a control). Anesthetized C57BL/6N mice were intranasally treated with 10 mg/kg (m.w. = 464 g/mol) SU0268 for 12 h before infection with 6 × 106 CFU of PA14.

Cell transfection

Lipofectamine RNAiMAX transfection reagent (13-778-075; Invitrogen, Carlsbad, CA) was diluted in 150 μl with Opti-MEM medium. Then, 150 μl mixture containing 150 μl Opti-MEM medium and 9 μl of small interfering RNA (siRNA) or 5 ng DNA was added to the diluted transfection reagent and incubated for 5 min at room temperature. Two hundred and fifty microliters of the mixture was added to each well. The knockdown efficiency was detected by Western blotting after 48 h transfection (Supplemental Fig. 4B).

MTT assay

Cells were stained with trypan blue, and the number of viable cells was quantified through a cell-counting plate. The concentration gradient of the SU0268 was added to the cells in a 96-well plate and incubated at 37°C/5% CO2 for the listed time. Twenty microliters of MTT solution (M6494; Thermo Fisher Scientific) (5 mg/ml, 0.5% MTT) was added to each well and continued to culture for 4 h. Stop solution was added to dissolve the formazan product. The cell viability was quantified by absorbance measurement at 560 nm using a spectrometric plate reader.

OGG1 inhibition in MH-S cells

MH-S cells (American Type Culture Collection) were grown in RPMI 1640 media (Life Technologies) supplemented with 10% FBS (Corning), 100 U/ml penicillin–streptomycin (Life Technologies) and 0.05 M 2-ME (Thermo Fisher Scientific) in a humidified incubator at 37°C with 5% CO2. For inhibitor treatment, media was supplemented with 1 μM SU0268 and replenished every 12 h for 36 h. For oxidative treatment, cells were treated with serum-free media containing 5 mM H2O2 and 100 μM Fe(NH4)2(SO4)2 for 1 h prior to harvesting. To prepare lysates, cells were collected in PBS by scraping, and the protocol for the CellLytic NuCLEAR Extraction Kit (Sigma-Aldrich) was used with Roche complete mini EDTA-free protease inhibitor tablets. Briefly, cells were grown to ∼90% confluency and harvested by scraping (∼5 × 107). Cells were rinsed twice with cold PBS and swelled in hypotonic lysis buffer for 15 min on ice (10 mM Tris [pH 7] with 1.5 mM MgCl2, 10 mM NaCl, 0.1 M DTT, and 1× protease inhibitor). Cells were lysed by repeated passage through a 25-gauge needle and the cytosolic fraction collected. Nuclear proteins were extracted from the nuclear pellet by shaking with a high salt nuclear extraction buffer for 60 min (20 mM HEPES [pH 7.9] with 1.5 mM MgCl2, 420 mM NaCl, 0.1 M DTT, 25% glycerol, and 1× protease inhibitor). The nuclear and cytosolic fractions were then combined, and total protein was determined by Bradford assay. OGG1 activity was measured in MH-S lysates (0.1 mg/ml) using the previously reported universal base excision reporter probe system (23). Briefly, lysates were incubated with an 8-oxoG–containing substrate (5 μM) and the glycosylase probe CCVJ1 (25 μM) with either SU0268 (1 μM) or a DMSO control for 4 h prior to measuring fluorescence. Percent activity represents the enzyme activity relative to the DMSO control with background subtracted (lysate and probe without substrate).

Measurement of 8-oxoG in DNA

MH-S cells were harvested by scraping (5 × 106) and rinsed twice with PBS. Genomic DNA was isolated using a Quick-DNA Miniprep Plus Kit (Zymo Research) according to the manufacturer’s instructions and concentration assessed by UV absorbance on a NanoDrop instrument (Thermo Fisher Scientific). DNA was melted at 95°C for 5 min, then rapidly chilled and digested with nuclease P1 (New England Biolabs) for 2 h at 37°C before a second heat treatment at 75°C for 10 min to inactivate the enzyme. A commercial ELISA kit for 8-oxoG determination (Abcam) was used according to the manufacturer’s instructions. Absorbance measurements were taken on an Infinite M1000 microplate reader (Tecan) and compared against a calibration curve. Meanwhile, the expression level of 8-oxo-dG was also assessed by using HT 8-oxo-dG ELISA Kit II (4380-096-K; Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions. Briefly, MH-S cells were treated with 2 μM SU0268 for 8 h and collected in 1.5 ml microtubes, washed two times with PBS, centrifuged at 10,000 × g/min for 10 s at 4°C, the supernatant discarded, and the DNA extracted from the above-collected cell pellets by using DNeasy Blood and Tissue Kit (69504; QIAGEN) according to the manufacturer’s instructions. Twenty-five microliters of 8-OHdG standards and clarified samples were added to each well (assay diluent buffer without 8-OHdG as background control), then 25 μl of anti 8-OHdG monoclonal solution, 50 μl of diluted Goat anti-Mouse IgG-HRP conjugate, and 50 μl of prewarmed TACS-Sapphire colorimetric substrate were added sequentially to all wells except blank wells and incubated in the dark for 15 min at 25°C. Reactions were stopped by adding 5% phosphoric acid. The absorbance was immediately read at 450 nm.

RNA isolation and quantitative PCR

RNA was extracted from lung tissues with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was obtained through reverse transcription reaction (Invitrogen). Genes were quantified by using real-time quantitative PCR (qPCR) reaction (Bio-Rad Laboratories, Hercules, CA). The PCR primers used in the reaction are listed in Supplemental Tables I and II.

Measurement of inflammatory cytokines

Cytokine concentrations were measured by an ELISA kit (eBioscience, San Diego, CA) in MH-S cells following the instruction of the manufacturer in 96-well plates (9018; Corning Costar). Briefly, plates were coated with 100 μl/well capture Ab in coating buffer and incubated overnight at 4°C. One hundred microliters of detection Ab with 1× ELISA/ELISPOT buffer was added to each well. Cytokine concentrations were determined with the corresponding detection of HRP-conjugated Abs. Optical absorbance was read at 450 nm and analyzed.

Western blotting

Mouse mAbs against β-actin (sc-47778) and HRP-labeled secondary Ab (sc-49395) were obtained from Santa Cruz Biotechnology. IL-1β (12242), TNF-α (5178), IL-6 (12912), cGAS (31659), p-TBK (5483), TBK (3504), IRF-3 (4302), p–IRF-3 (79945), and guanine nucleotide exchange factors (GEFs; 4076) were purchased from Cell Signaling Technology (Danvers, MA); and KRAS (sc-30), NF-κB p65 (sc-8008), and ERK1 p44 (sc-271291) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cells and homogenized lung tissue were lysed in radioimmunoprecipitation assay buffer containing protease inhibitor (Thermo Fisher Scientific), then were denatured in 100°C for 10 min, separated by electrophoresis on 12% SDS-PAGE gels, and then transferred to nitrocellulose transfer membranes (GE Amersham Biosciences, Pittsburgh, PA). Membranes were incubated with primary Abs overnight, then were incubated with corresponding secondary Abs conjugated to HRP (Santa Cruz Biotechnology). Finally, the relative expression levels of protein were detected using ECL reagents (Santa Cruz Biotechnology) and were quantified by Quantity One software (Bio-Rad Laboratories).

Immunostaining

Cells were cultured overnight in 24-well plates containing slides. Cells were fixed with 4% paraformaldehyde (PBS) for 15 min and washed with PBS three times for 3 min each time. Three hundred microliters of 0.5% Triton X-100 (diluted in PBS) was added to each well and permeabilized for 20 min at room temperature. Then, 5% BSA was added on the slide and blocked at room temperature for 30 min. Primary Ab that was diluted in 1% BSA was added in the amount of 200 μl/well, and slides were put into the wet box and incubated overnight at 4°C. Then the slides were incubated with the appropriate fluorescein-conjugated secondary Abs in the dark. For detection of released mtDNA, MH-S cells were marked by transfected plasmids MSCV-IRES-puromycin-TFAM-mNeonGreen (coexpression of the mtDNA-binding protein transcription factor A, which was tagged with the GFP variant mNeonGreen and used to track mtDNA) and MSCV-IRES-hygromycin-TOMM20 Halo (TOMM20) (24) (gift from B.T. Kile, Department of Medical Biology, University of Melbourne; used to track mitochondria), and then DAPI (Sigma-Aldrich) was used to stain the nucleus for 10 min in the dark at room temperature. The coverslips were mounted on slides with VECTASHIELD mounting medium. The images were captured with a Zeiss Meta 510 confocal microscope (Carl Zeiss, Oberkochen, Germany). NF-κB p65 (sc-8008) Abs were purchased from Santa Cruz Biotechnology, and TNF-α (11948), IL-6 (12912), cGAS (31659), and IRF-3 (4302) Abs were purchased from Cell Signaling Technology.

NBT assay

MH-S cells were placed in 96-well plates in serum-containing complete medium and were attached at 37°C for 4 h. One microgram per milliliter NBT dye (298-83-9; Sigma-Aldrich, St. Louis, MO) was added to each well and incubated at 37°C for 1 h until the color changed from yellow to purple formazan. One hundred microliters of stop solution (10% DMSO and 10% SDS in 50 mM HEPES buffer) was added to each well. The plate was placed at room temperature overnight to completely dissolve the formazan, and the OD value read at 560 nm. Each experiment was performed in triplicate.

Dihydrodichlorofluorescein diacetate (H2DCF-DA) assay

MH-S cells were transfected with 5 μM H2DCF-DA staining solution (Molecular Probes, Carlsbad, CA) in a 96-well plate and incubated at 37°C for 30 min, and fluorescence was measured by a fluorescence plate reader (BioTek Instruments, Winooski, VT) using 485 nm excitation and 528-nm emission filter. The images were captured on a Zeiss Meta 510 confocal microscope (Carl Zeiss).

Comet assay

The comet assay was performed by using the CometAssay HT kit (4252-040-K; Trevigen) according to the manufacturer’s instructions. Briefly, MH-S cells were treated with SU0268, then were infected with PA14; cells were then harvested and resuspended in ice-cold PBS. Cells (1 × 105/ml) were combined with low melting agarose (kept in 37°C water bath for at least 20 min to cool) at a ratio of 1:10 (v/v). Immediately afterward, 30 μl was pipetted onto the 20-well CometSlide. Slides were laid flat at 4°C in the dark for 10 min, immediately immersed with lysis solution, and incubated overnight at 4°C, then were washed with unwinding solution (pH > 13). Alkaline electrophoresis was performed at 21 V for 30 min, slides were immersed in the diluted staining solution containing with 50 μl SYBR Gold for 30 min in the dark, and the images were captured with a Zeiss Meta 510 confocal microscope (Carl Zeiss).

Assessment of mitochondrial membrane potential

MH-S cells were collected in the 96-well plate. Ten microliters of the JC-1 staining solution was added and incubated in the incubator for 30 min. The plate was then centrifuged at 400 × g for 5 min at room temperature, and the supernatant was discarded. Then, 200 μl of assay buffer was added to each well, and fluorescence values of cells were analyzed with a fluorescence plate reader. Strong fluorescence intensity with emission at 535 and 595 nm is typical for healthy cells but at 485 and 535 nm for unhealthy cells. Triplicates were performed for each sample and control (25). The images were captured with a Zeiss Meta 510 confocal microscope (Carl Zeiss).

Extraction and quantification of mtDNA release in cytoplasm

MH-S and J744A.1 cells (1 × 106) were collected and then were washed once with 1× Dulbecco’s PBS. Then, 1% NP-40 (100 μl) was added to each well, and the cells were scraped. Lysates were placed into prelabeled microcentrifuge tubes and incubated on ice for 15 min. Lysates were spun at 13,000 rpm for 15 min at 4°C to pellet the insoluble fraction. Supernatant (the cytosolic fraction) was transferred to a new tube, and the pellet was discarded. The supernatant was used in the next step to extract cytosolic mtDNA by using DNeasy Blood and Tissue Kit (69504; QIAGEN) according to the manufacturer’s instructions. Finally, the expression levels were quantified by qPCR. The following primers were used: cytochrome C oxidase I (mtDNA gene) and housekeeping gene (18S rRNA-encoding DNA [rDNA]) (26, 27). The PCR primers used in the manuscript are listed in Supplemental Table I.

Assessment of apoptosis

The cells (cell numbers > 1 × 105) were collected after treatment with SU0268 for 8 h, followed by infection with PA14 for 2 h, washed twice with PBS, resuspended, and centrifuged for 4 min at 3000 × g/min. A total of 1.5 μM propidium iodide staining (V13242; Thermo Fisher Scientific) solution was added and incubated in the dark for 30 min at room temperature. Apoptosis was detected by flow cytometry.

Immunoprecipitation

MH-S cells were carefully washed with prechilled PBS two times. Prechilled radioimmunoprecipitation assay lysis buffer (50 mM [pH 7.4] Tri-HCl, 15 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1.25% Triton X-100, and protease inhibitor) was added, and then MH-S cells were collected to sterilized 1.5-ml Eppendorf tubes using a prechilled scraper, then cells were centrifuged at 14,000 × g/min for 15 min at 4°C, and the supernatant was incubated with the magnetic agarose beads containing the Ab overnight (Dynabeads M-280 sheep anti-rabbit; Invitrogen). Cells were washed twice with low-salt wash buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, and 0.05% NP-40) and then were divided into two equal parts, one for detecting immunoprecipitation efficiency by Western blotting. Another magnetic bead sample was added with 200 μl of DNA decross-linking solution (10 mM Tris, 1 Mm EDTA, and 0.65% SDS), incubated at 65°C for 16 h, and then proteinase K (20 mg/ml) was used to digest protein by incubating for 2 h at 37°C. The DNA that bound to the cGAS protein was extracted through the mixture of phenol, chloroform, and isoamyl alcohol (25:24:1) (Thermo Fisher Scientific) and then was precipitated in 75% ethanol. Finally, the DNA was dissolved in the nuclease-free water and quantified by qPCR by using cytochrome C oxidase I (mtDNA gene) and housekeeping gene (18S rDNA) (27); the PCR primers used in the reaction are listed in Supplemental Table I.

mtDNA deletion and replenishment

For the analysis of how mtDNA manipulates type I IFN responses, mtDNA knockout and replenishment assay was performed by using Lip RNAiMAX Reagent and Pierce Protein Transfection Reagent Kit (Waltham, MA) according to the manufacturer’s instructions. Briefly, J744.1A macrophages were cultured to 80% density in RPMI 1640 medium supplemented with 10% FBS and then were induced for 2 wk to remove mtDNA by adding 1000 ng/ml ethidium bromide, 25 μg/ml uridine, and 1 mM pyruvate (medium was changed once per day). To analyze whether mtDNA was completely knocked out, mtDNA copy numbers were determined by qPCR using primers for cytochrome C oxidase I (mtDNA gene) and housekeeping gene (18S rDNA) (Supplemental Fig. 4C). DNase I that was used to digest exogenously imported mtDNA was transfected into cells by using Pierce Protein Transfection Reagent Kit (89850; Thermo Fisher Scientific) according to the manufacturer’s instructions, and mtDNA, which was obtained from the cytosol by using DNeasy Blood and Tissue Kit according to the manufacturer’s instructions, was replenished into J744.1A macrophages that had deleted mtDNA by using Lip RNAiMAX Reagent as previously described (28).

Bacterial burden assay

Animal blood was obtained by cardiac blood sampling on the sacrificed mouse, lung tissue was homogenized with PBS, BALF was obtained through alveolar lavage, and all samples were diluted to different gradients in PBS before being evenly distributed in lysogeny broth dishes. The dishes were cultured in a 37°C incubator overnight, and the number of bacteria was counted. Each experiment was performed in triplicate.

Histological analysis

Lung tissues were fixed in 10% formalin (Sigma-Aldrich) for 48 h in 4°C and then embedded in paraffin using a routine histologic procedure. H&E staining was carried out according to the standard staining procedure (26).

Flow cytometry

BALF was obtained through alveolar lavage, blood samples were obtained through cardiac blood sampling, and erythrocytes were removed through the cell lysate. Lung tissues were homogenized with PBS and digested with collagenase. They were washed three times with PBS, then resuspended in 500 μl PBS. Two hundred microliters of CD45, CD11b, and MHC class II (MHCII) Ab (BioLegend, San Diego, CA) with PBS (1:1000) were added to the groups of cells separately; CD45+/CD11b+ was used to mark myeloid progenitors, and CD11b+/Gr1+/MHCII+ was used to mark neutrophils. Cells were incubated in the dark for 30 min at room temperature, washed two times with PBS, and then resuspended in 1 ml PBS. The cells were filtered by using a 40-μm filter and were tested by flow cytometry (29).

Quantification and statistical analysis

Data were presented as mean ± SEM determined from biological triplicates. Statistical analysis was performed with GraphPad (GraphPad Software, La Jolla, CA). Results in Figs. 1, 4A, and 5A were analyzed by Student t test compared with control group. Results in Figs. 2A, 2B, 3, 4B, 5B, 5D–F, and 6B, 6D, and 6E were analyzed by one-way ANOVA plus Tukey post hoc test; data in Fig. 6A were analyzed by Kaplan–Meier survival analysis. Statistically significant differences are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

SU0268 potently inhibits the activity of OGG1 and decreases levels of proinflammatory cytokines in vitro

To determine the intrinsic cytotoxicity of SU0268 on mouse alveolar macrophage (MH-S) cells, we conducted MTT assays evaluating cell viability treated with different concentrations of the SU0268 (50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78, and 0.39 μM) for 24 h. The data of Fig. 1A showed that SU0268 possessed some toxicity to MH-S cells with IC50 at 14.7 μM, and the toxicity is higher than in HEK293 and HeLa cell lines (15), which may be related to the different tolerance to inhibitors (SU0268) between human and mouse cell types. We further tested doses lower than IC50 to determine the optimal concentration for blocking OGG1 activity as 2 μM (Fig. 1B), which did not influence cell viability over an extended time (Fig. 1C). We thus chose 2 μM SU0268 for further study. To determine whether SU0268 can inhibit the activity of murine OGG1 from MH-S cells, we used a previously published fluorescence assay specific for OGG1 activity (23); the data showed when lysates were supplemented with SU0268 (1 μM) and observed that OGG1 activity was dampened to ∼15% relative to the DMSO control, and the enzyme activity of OGG1 was strongly suppressed by SU0268 at 1 μM (Fig. 1D). Meanwhile, SU0268 also mildly inhibited the expression level of OGG1 by SU0268 at 2 μM for varied times (Supplemental Fig. 1A, 1B). Further experiments carried out by incubating MH-S cells with SU0268 confirmed that the inhibitor increased levels of 8-oxo-dG in DNA, as expected after suppressed excision of this lesion (Fig. 1E, Supplemental Fig. 1C).

FIGURE 1.
FIGURE 1.

The chemical inhibitor SU0268 suppresses OGG1’s activity and inflammatory response in cell culture. (A) Cell viability detected by MTT assay. MH-S macrophages were treated with SU0268 (50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78, and 0.39 μM) for 24 h and IC50 was calculated with GraphPad 7.0. (B) Cell viability evaluated after treating cells with SU0268 at 0, 2, 4, 8, and 16 μM, respectively. (C) Cell viability was further evaluated by MTT at 2 μM for indicated times. (D) Endogenous OGG1 activity in lysates prepared from MH-S cells (0.1 mg/ml total protein) was measured using a previously described fluorescence assay (23). (E) MH-S cells were cultured in the presence of SU0268 (1 μM) for 36 h or treated with H2O2 (5 mM) and Fe2+ (100 μM) for 1 h. Total DNA was then isolated and digested with nuclease P1 prior to measurement of 8-oxoG using a commercial 8-hydroxy 2-deoxyguanosine ELISA kit. The anti-inflammatory effect of SU0268 was evaluated by quantifying the expression level of TNF-α, IL-1β, and IL-6 by using ELISA (F and G) Western blotting after treatment with SU0268 (2 μM) for 0, 4, 8, and 12 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Recent studies have demonstrated that inhibition of the expression of OGG1 can effectively inhibit the inflammatory response (7, 16). To further analyze the role of SU0268 as an anti-inflammatory agent in MH-S cells, the expression levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β at different time points were evaluated using ELISA (Fig. 1F) and Western blotting (Fig. 1G) after incubation with 2 μM SU0268 (MH-S cells can spontaneously produce inflammatory factors, albeit at much lower levels) (26, 30). Basal expression of inflammatory mediators was suppressed by SU0268, and the anti-inflammatory activity appeared to be strongest at 4 and 8 h posttreatment. Altogether, these data showed that SU0268 potently inhibits the activity of OGG1 and downregulates levels of proinflammatory cytokines in vitro.

SU0268 regulates innate inflammation through KRAS–ERK1–NF-κB axis

To further analyze the role of SU0268 in P. aeruginosa infection, the anti-inflammatory activity of SU0268 in PA14 infection was quantified by qPCR (Fig. 2A), ELISA (Fig. 2B), and Western blotting (Supplemental Fig. 2A). At rest, the NF-κB dimers are sequestered in the cytoplasm by binding with IκBs (31). NF-κB is activated upon LPS or bacterial challenge and is then freed to enter the nucleus to bind promoter regions of certain genes to initiate the inflammatory responses (32–34). Next, activation of NF-κB and the expression level of proinflammatory cytokines (TNF-α and IL-6) were evaluated by immunofluorescence (Fig. 2C, Supplemental Fig. 2B). Furthermore, OGG1, as a DNA repair enzyme, initiates excessive inflammatory responses by activating the OGG1–BER–KRAS–ERK1/2–NF-κB circuit when bacterial infection induces DNA base lesions (7, 35, 36); the activation of KRAS is regulated by GEFs (37). In this study, we found that OGG1 regulation of inflammation is related to the GEFs/KRAS–ERK1–NF-κB axis, which was drastically downregulated by SU0268 in cell culture (Fig. 2D). Together, these results suggest that SU0268 decreases innate inflammation by inhibiting the GEFs/KRAS–ERK1–NF-κB axis upon P. aeruginosa infection.

FIGURE 2.
FIGURE 2.

SU0268 inhibits inflammatory responses in MH-S cells via OGG1– GEFs/KRAS–NF-κB axis. MH-S cells were treated with SU0268 (2 μM) for 8 h (the control group received an equal amount of 100% ethanol), then infected with PA14 for 2 h. Anti-inflammatory activity of SU0268 was analyzed by measuring the expression of TNF-α, IL-1β, and IL-6 by qPCR (A) and ELISA (B). The activation of NF-κB was detected by immunofluorescence (C) (arrows indicating cytoplasmic NF-κB staining; the typical cells were enlarged five times and placed on the bottom). (D) The protein expression levels of OGG1–GEFs/KRAS–NF-κB axis were determined by Western blotting. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

OGG1 inhibitor regulates the release of mtDNA

ROS production has been linked to various cellular signaling pathways (38). The expression levels of ROS are documented to increase upon exposure to bacterial infection (39). Increased ROS-induced DNA damage can be repaired by OGG1 through the DNA BER pathway (OGG1–BER) (40). However, the levels of ROS-induced DNA damage have not been decreased but increased by generating repair intermediates (41). To investigate the impact of ROS on DNA damage during P. aeruginosa infection, the amount of ROS released was measured using NBT (Fig. 3A) and H2DCF assay (Fig. 3B, Supplemental Fig. 3A). Concomitantly, the degree of DNA damage was assessed by the comet assay (Fig. 3C), and results demonstrate increased ROS and DNA damage in MH-S cells by P. aeruginosa. Significantly, treatment with SU0268-attenuated DNA damage compared with P. aeruginosa infection, which is consistent with lowered levels of base excision and decreased levels of DNA strand cleavage. Mitochondria play an important role in regulating the innate immune response to infection and DNA damage (42). The accumulation of DNA damage initiates mitochondrial dysfunction (43). To investigate the role of mitochondria in the regulation of P. aeruginosa infection, we examined mitochondrial potential using the JC-1 fluorescence assay and found that SU0268 significantly restored the infection-mediated mitochondrial potential (Fig. 3D, Supplemental Fig. 3B). mtDNA is critical for maintaining sufficient cellular energy required for cell development and function, in addition to regulating a variety of immune responses (44). mtDNA maintenance defects, which are caused by mtDNA synthesis disorders, will further trigger insufficient mtDNA-encoded protein synthesis and energy production, resulting in organ dysfunction (45, 46). We used qPCR (Fig. 3E) and immunofluorescence (Supplemental Fig. 3C) to detect released mtDNA that was increased upon P. aeruginosa infection and further augmented by incubation with SU0268. Collectively, these observations strongly imply that this OGG1 inhibitor regulates the mitochondrial immune response by regulating the release of mtDNA.

FIGURE 3.
FIGURE 3.

SU0268 promotes mtDNA release into the cytoplasm after ROS-induced DNA damage in response to P. aeruginosa infection. MH-S cells were treated with SU0268 (2 μM) for 8 h (the control group received an equal amount of 100% ethanol), then infected with PA14 for 2 h. The ROS products were measured by NBT assay (A) and H2DCF assay (B) according to the manufacturer’s instructions. (C) DNA strand break damage was detected by comet assay, and the tail length of damaged DNA was measured with confocal laser scanning microscopy. (D) Mitochondrial membrane potential was assessed by JC-1 fluorescence assay. (E) mtDNA was extracted, and mtDNA copy numbers were analyzed by qPCR. *p < 0.05, **p < 0.01, ***p < 0.001.

OGG1 inhibitor facilitates mtDNA release by manipulating BAK/BAK macropores-induced apoptosis

To further characterize the critical role of an OGG1 inhibitor in modulating the mitochondrial immune response by regulating the release of mtDNA in P. aeruginosa infection, a qPCR microarray was executed to screen 60 genes involved in mtDNA release and mitochondrial function after treatment with SU0268 for 8 h, then infection with PA14 for 2 h, with PA14 infection group lacking SU0268 as control (Fig. 4A) (gene primers are shown in Supplemental Table II). We found that the expression of Bak and Bax was significantly increased. Bak/Bax, as a proapoptotic factor, mediates cell clearance programming and tissue homeostasis to prevent a potentially damaging inflammatory or immune response (24). We hypothesized that BAK/BAX may receive a signal from apoptotic mitochondria, which may be associated with DNA damage. We found that cell apoptosis was increased by the OGG1 inhibitor SU0268 (Fig. 4B). Meanwhile, cytochrome C loss has also been shown to manipulate the release of mtDNA by mitochondrial apoptosis (24, 47). To firmly determine whether increased the release of mtDNA is associated with mitochondrial apoptosis, immunofluorescence and Western blotting were also performed to detect released cytochrome C from mitochondria. We found that cytochrome C loss was significantly increased following infection and could be further increased by the OGG1 inhibitor (Fig. 4C). These results together suggest that the OGG1 inhibitor facilitates mtDNA release by modulating BAK/BAX macropore-induced apoptosis.

FIGURE 4.
FIGURE 4.

SU0268 promotes mtDNA release dependent on the BAK/BAX axis. MH-S cells were treated with SU0268 (2 μM) for 8 h (the control group received an equal amount of 100% ethanol), subsequent infection with PA14 for 2 h. (A) Sixty genes involved in mtDNA release and mitochondrial function were quantified by using qPCR microarray, heatmaps, and red dots in scatter plot are used to indicate differential expression of genes (more than 2-fold change and p < 0.05 by Student t test, respectively; each color of 1–60 represents the expression of one gene; primers are shown in Supplemental Table II). (B) Cell apoptosis was measured by flow cytometry; ABT737, as a positive control, has been identified to cause apoptosis (77). (C) Released cytochrome C was detected by immunofluorescence (arrows indicating typical released cytochrome C) and Western blotting (right panel). *p < 0.05, **p < 0.01, ***p < 0.001.

Increased release of mtDNA activates the cGAS–TBK–IRF3–IFN-β pathway by increasing binding with cGAS to influence type I IFN responses

Research has shown that Ogg1 knockout mice have increased oxidized mtDNA in macrophages (48). It remains unknown how mtDNA participates in immune regulation during P. aeruginosa infection. To gain further insights into the role of mtDNA in regulating inflammatory response, we used RT2 Profiler PCR Cancer Inflammation and Immunity Cross-talk array to identify key regulatory genes (330231 PAMM-181Z; QIAGEN) (Fig. 5A). The results show that the Stat1 levels were highly activated. Tyrosine kinase 2 (TYK2) and JAK1 enable the phosphorylation of STAT1/STAT2, which activates the transactivation of multiple genes, including cGAS (49). It has been widely reported that mtDNA facilitates the activation of the cGAS–TBK–IRF3 circuit (50). We hypothesized that elevated released mtDNA activates the cGAS pathway upon P. aeruginosa infection. Therefore, the cGAS signaling pathway proteins were measured by Western blotting (Fig. 5B), and nuclear transfer of IRF3 was also augmented as detected by immunofluorescence (Supplemental Fig. 4A). To clarify the mechanism of the mtDNA-driven cGAS pathway, colocalization of mtDNA and cGAS was revealed by immunofluorescence (Fig. 5C). Next, a cGAS Ab was used to pull down mtDNA under different processing conditions using immunoprecipitation assay, and mtDNA was coprecipitated by cGAS Abs, which was quantified by qPCR (Fig. 5D). These data strongly support that increased release of mtDNA activated the cGAS pathway by increasing binding to cGAS. Activation of the cGAS pathway is critical for the generation of type I IFNs (51). Certainly, IFN is essential for host defense against bacterial infection by modulating innate immune responses (52). Mice exhibited significantly improved clearance of P. aeruginosa after being pretreated with the activator of IFN-β (53). Next, we examined the release of IFN-β in MH-S and J744.1A cells by ELISA (Fig. 5E, 5F), and the results revealed that the OGG1 inhibitor elicited increased expression of IFN-β. Notably, this situation was reversed after knocking down cGAS with siRNA (Fig. 5E); the cGAS knockdown efficiency was detected by Western blotting after 48-h transfection (Supplemental Fig. 4B). Meanwhile, to further confirm that mtDNA regulation the release of IFN-β is through cGAS-related signal pathway, mtDNA deletion and replenishment assays were performed in J744.1A macrophages without mtDNA (Supplemental Fig. 4C, detailed in Materials and Methods); the results revealed that replenishment mtDNA increased expression of IFN-β. However, there is no significant difference when knocking down cGAS with siRNA (Fig. 5F). These results strongly suggest that mtDNA regulation of the type I IFN responses was through the cGAS signaling pathway. Consistently, these experiments demonstrate that increased release of mtDNA activates the cGAS–TBK–IRF3–IFN-β pathway by increasing binding with cGAS to manipulate type I IFN responses.

FIGURE 5.
FIGURE 5.

Increased release of mtDNA activates the cGAS pathway by directly binding cGAS protein to manipulate type I IFN responses. MH-S cells were treated with SU0268 (2 μM) for 8 h (the control group received an equal amount of 100% ethanol), subsequent infection with PA14 for 2 h. (A) RT2 Profiler PCR inflammation and immunity cross-talk array was used to identify key regulatory genes (330231 PAMM-181Z; QIAGEN). Heatmaps and red dots in scatter plots are used to indicate differential expression of genes (more than 2-fold change and p < 0.05 by Student t test), respectively, each color of A01-G12 represents the expression of one gene. (B) The expression levels of proteins (cGAS, p-TBK, TBK, p-IRF3, and IRF-3) were quantified through Western blotting. (C) Colocalization of mtDNA and cGAS was detected by immunofluorescence (arrows indicating typical colocalization of mtDNA and cGAS). (D) Mitochondria DNA that was bound to cGAS was analyzed by immunoprecipitation, then quantified by qPCR. (E) MH-S cells and J744.1A were transfected with cGAS siRNA for 48 h (negative siRNA as control) by using Lip RNAiMAX Reagent, then treated with SU0268 for 8 h (pure ethanol as control), followed by PA14 infection for 2 h. The expression level of IFN-β was determined by ELISA (42400-1; R&D Systems) according to the manufacturer’s instructions. (F) Mitochondria DNA deletion and replenishment assays were performed in J744.1A cells as previously described (26). J744.1A cells that deleted mtDNA were transfected with cGAS siRNA (negative siRNA as control) for 36 h and subsequently treated with PBS or DNAase I or heated DNAase I for 4 h, then mtDNA was replenished for 12 h in pretreated PBS group by using Lip RNAiMAX Reagent, and the expression level of IFN-β was analyzed by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001.

Small-molecule inhibitor of OGG1 significantly inhibits inflammatory responses and mitigates P. aeruginosa infection

To gain deeper insights into the role of OGG1 inhibitor–mediated inflammatory and mtDNA-mediated type I IFN responses in P. aeruginosa infection in vivo in parallel with in vitro, we performed a survival curve to explore the prevention and therapeutic effects of SU0268 in MH-S cells and C57BL/6N mice. The results showed that pretreatment with SU0268 increased survival rates compared with controls without SU0268 pretreatment in MH-S cells and C57BL/6N mice (Fig. 6A, Supplemental Fig. 5A). Moreover, SU0268 also showed therapeutic activity after the MH-S cells, and animals were infected by the PA14 bacterium (Supplemental Fig. 5B, 5C). In a separate study, mouse pharmacokinetic data for SU0268 showed half-life (t1/2) ∼4.0 ± 0.7 h as performed. Next, bacterial burdens were quantified by CFUs in the lung, blood, and BALF (Fig. 6B, Supplemental Fig. 5D), which showed that the burden of PA14 was significantly decreased in the mice treated with SU0268. Furthermore, we also found significant improvement in lung tissue damage in mice by immunohistochemistry staining (Fig. 6C) and significantly decreased expression levels of TNF-α, IL-6, and IL-1β in the presence of the inhibitor (Supplemental Fig. 5E). Conversely, the expression level of IFN-β was upregulated in the mouse primary BMDM cells after treatment with SU0268 for 8 h, followed by infection with PA14 for 2 h (Fig. 6D). Neutrophils play essential roles in the clearance of bacteria and viruses (54). However, excessive recruitment of neutrophils significantly aggravates the injury of lung tissue (55, 56). Next, we examined the number of neutrophils in mouse BALF, blood, and lung by staining with CD11b+/Gr1+/MHCII+ Abs via flow cytometry (Fig. 6E). We found the myeloid progenitor cells (CD45+/CD11b+) and neutrophils (CD11b+/Gr1+/MHCII+) to be significantly increased in PA14 infection but significantly decreased with prior treatment of SU0268. Consistent with previous observations, this result supports the conclusion that the small-molecule OGG1 inhibitor regulates inflammatory response and mtDNA-mediated type I IFN release to mitigate bacterial infection during P. aeruginosa infection in vivo and in vitro, as shown in Fig. 7.

FIGURE 6.
FIGURE 6.

Small-molecule inhibitor of OGG1 significantly downregulates proinflammatory responses and mitigates bacterial infection. Mice treated with SU0268 (10 mg/kg) for 12 h and control mice without SU0268 treatment (n = 9) were intranasally challenged with PA14 at 6 × 106 CFU (uninfected WT and SU0268 as a control) and observed up to 9 d. (A) The survival test is represented by Kaplan–Meier survival curves, and the results showed that pretreatment with SU0268 increased survival rates compared with PA14 infection without SU0268 pretreatment (p = 0.0925; 95% confidence interval, n = 9 for each group). (B) Colony formation unit assay was used to count the number of bacteria in the lung, blood, and BALF at 9 d postinfection. (C) H&E staining was used to assess lung injury in mice (photograph is magnified 20 times). (D) ELISA was performed to determine the expression level of IFN-β in the mouse primary BMDM cells. (E) Ten thousand cells that were stained by indicated Abs were collected, the percentages of the myeloid progenitor cells (stained CD45+/CD11b+ cells/total cells) and neutrophils (stained CD11b+/Gr1+/MHCII+ cells/total cells) in mouse BALF, blood, and lung were detected by flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 7.
FIGURE 7.

Schematic illustration of the signaling pathways in which small-molecule inhibitor of OGG1 orchestrates inflammatory and mtDNA-mediated type I IFN responses. Normally, PA14 infection triggers increased ROS production and DNA damage, which moderately activates OGG1’s damage repair function and induces an excessive inflammatory response by regulation of the KRAS–ERK1–NF-κB axis. Inhibiting the enzymatic activity of OGG1 decreases DNA inflammatory responses. Furthermore, the small-molecule inhibitor of OGG1 elicits massive mtDNA release into the cytoplasm, which then initiates IFN-β responses by the cGAS–TBK–IRF3–IFN-β circuit. Ultimately, elevated IFN-β mitigates P. aeruginosa infection and spread.

Discussion

In this study, we investigated the role of the OGG-1 inhibitor SU0268 in P. aeruginosa infection, which showed that pharmacological inhibition of OGG1 in place of a knockout strategy decreased the expression of proinflammatory factors, allowing the approach to be applicable in clinical scenarios. Importantly, our data elaborated a new mechanism whereby a small-molecule OGG1 inhibitor decreases inflammatory and mtDNA-mediated type I IFN responses to inhibit bacterial spread and improve susceptibility after P. aeruginosa infection. Our research provides theoretical support for a small-molecule inhibitor as a potential anti-infective drug to decrease bacterially induced inflammation.

Stimulation of the external environment, such as bacterial or viral infections, will result in dysregulation of ROS generation, which will cause DNA damage (57). OGG1, which acts as a DNA BER enzyme, is crucial for modulation of the coordination between innate and adaptive immunity through regulating oxidative stress and immune responses (58). In the BER pathway, OGG1 binds to 8-oxoG adducts to form an OGG1/8-oxoG complex, which functions as a prototypic GEF (59). The Rho family of GTPases, a subfamily of the Ras superfamily, controls multiple cellular processes, including gene expression, microtubule dynamics and membrane transport, and are activated by GEFs (60–62). It has previously been reported that OGG1 increased activation of Rho-GTPase in oxidatively stressed cells (63). GEF-H1 (a RhoA activator, as a GEF for the Rho small GTPase proteins) has been well documented to act as an integrator of microtubule and actin dynamics in diverse cell functions (64). GEF-H1 is also shown to promote RAS-driven MAPK via modulating KSR-1 phosphorylation (65). OGG1 initiates inflammatory responses by activating the OGG1/KRAS/NF-κB pathway during bacterial infection or ROS-induced DNA damage (7, 41). Our data demonstrate that SU0268 decreases the inflammatory responses through the GEFs/KRAS–ERK1–NF-κB axis, which may be related to decreased OGG1-BER activity and thereby downregulating GEFs expression. This will inhibit Ras activation by regulating the exchange of GDP to GTP and decreasing expression of proinflammatory genes and innate immune responses (66). The extent of bacterial replication may be influenced by host antibacterial immune responses, including inflammatory responses (67). Meanwhile, anti-inflammatory reaction can suppress the growth of bacteria and reduce the burden of pathogenic damage during infection (9, 68). Together, our data showed that OGG1 inhibitor SU0268 decreased the inflammatory response through the GEFs/KRAS–ERK1–NF-κB axis, which may help mitigate P. aeruginosa infection.

The mouse strains lacking Ogg1 (Ogg1−/−) or Nth1 (Nth1−/−) have been reported to have an increase in the copy numbers of mtDNA, which may be related to the decrease in the repair function of OGG1 (69). mtDNA is a small circular dsDNA that localizes in the mitochondrial matrix. mtDNA functions as a signaling platform to facilitate antibacterial immunity and regulating antimicrobial signaling by activating different kinds of molecular pathways upon exposure to bacteria or viruses (59, 61, 62). mtDNA is released into the cytoplasm through BAK- and BAX-mediated mitochondrial apoptosis, where it can be recognized by DNA sensor cGAS and incite innate immunity responses through the cGAS/STING signaling (63), increasing type I IFN secretion (42). Our study demonstrated that OGG1 inhibitor (SU0268) increased the release of mtDNA into the cytoplasm, where it activates the cGAS–TBK–IRF3–IFN-β circuit and triggers type I IFN responses.

Type I IFNs as pleiotropic cytokines are essential for host defense against viral and bacterial infection by modulating innate and adaptive immune responses (70), which can be activated by the cGAS/TBK/IRF3 signaling pathway (71, 72). Activated type I IFNs play a decisive role in the inhibition of bacterial replication and regulation of anti-inflammatory responses to protect against tissue damage–induced mortality (51). Mechanistically, type I IFN inhibits the acute phase expression of TNF-α and IL-1β cytokines by upregulating the anti-inflammatory cytokine expression of IL-10 and IL-27 (73). IFNAR1- or NOD1-deficient mice that cannot induce the expression of type I IFN in an IRF7-dependent manner fail to restrict Helicobacter pylori proliferation and aggravated lung injury (74). Our analysis revealed that increased IFN-β secretion by mtDNA/cGAS/TBK/IRF3 axis mitigates P. aeruginosa infection. Together, our data illustrated that the OGG1 inhibitor SU0268 may regulate inflammatory responses to attenuate P. aeruginosa infection by activating mitochondrial immune response through an mtDNA/cGAS/TBK/IRF3/IFN-β axis. Other acute clinical lung infections are also exacerbated by an induced inflammatory response. For example, patients with confirmed coronavirus disease (COVID-19) show elevated levels of cytokines including IL-6, and it has been proposed that agents that lower IL-6 signaling may be an effective therapeutic approach (75, 76). Given that SU0268 inhibits IL-6 expression and inflammatory responses in P. aeruginosa infection, it suggests that further studies of this compound in models of viral lung infections, such as those caused by SARS-CoV-2, may also be warranted.

In summary, we have investigated the role of OGG1 inhibitor SU0268 in P. aeruginosa infection, which shows significant inhibition of inflammatory responses and mitigates P. aeruginosa (PA14) infection. Mechanistically, the small-molecule inhibitor of OGG1 orchestrates inflammatory and mtDNA-mediated type I IFN responses to regulate anti-inflammatory responses and attenuates bacterial infection by inhibiting the activity of OGG1 in excision of 8-oxoG and activating the mtDNA–cGAS–TBK–IRF3–IFN-β circuit. These results suggest that OGG1 may indirectly regulate the expression of IFN-β to mediate adaptive immunity, which provides novel, to our knowledge, insights for the future development of OGG1 inhibitors to treat bacterial infection.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

Immunofluorescent, histological, and flow cytometry images were captured at the University of North Dakota Imaging Core and Flow Cytometry Core with assistance from Dr. Abrahamson, Dr. DeMontigny, and Dr. Adkins.

Footnotes

  • This work was supported by National Institutes of Health Grants R01 AI109317-01A1, R01 AI138203, and AI097532-01A1 to M.W., CA217809 to E.T.K., P20 GM113123 to M.W. and the University of North Dakota Imaging Core, and P20 GM103442 to the University of North Dakota Flow Cytometry Core. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BALF
    bronchoalveolar lavage fluid
    BER
    base excision repair
    BMDM
    bone marrow–derived macrophage
    cGAS
    cyclic GMP–AMP synthase
    GEF
    guanine nucleotide exchange factor
    H2DCF
    dihydrodichlorofluorescein
    MHCII
    MHC class II
    OGG1
    8-oxoguanine DNA glycosylase-1
    8-oxoG
    8-oxo-7,8-dihydroguanine
    PA14
    P. aeruginosa reference strain 14
    qPCR
    quantitative PCR
    rDNA
    rRNA-encoding DNA
    ROS
    reactive oxygen species
    siRNA
    small interfering RNA.

  • Received December 31, 2019.
  • Accepted August 17, 2020.

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