Exogenous 8-Hydroxydeoxyguanosine Attenuates PM2.5-Induced Inflammation in Human Bronchial Epithelial Cells by Decreasing NLRP3 Inflammasome Activation

Particulate matter 2.5 (PM2.5) induces lung injury by increasing the generation of reactive oxygen species (ROS) and inflammation. ROS aggravates NLRP3 inflammasome activation, which activates caspase-1, IL-1β, and IL-18 and induces pyroptosis; these factors propagate inflammation. In contrast, treatment with exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases RAC1 activity and eventually decreases dinucleotide phosphate oxidase (NOX) and ROS generation. To establish modalities that would mitigate PM2.5-induced lung injury, we evaluated whether 8-OHdG decreased PM2.5-induced ROS generation and NLRP3 inflammasome activation in BEAS-2B cells. CCK-8 and lactate dehydrogenase assays were used to determine the treatment concentration. Fluorescence intensity, Western blotting, enzyme-linked immunosorbent assay, and immunoblotting assays were also performed. Treatment with 80 μg/mL PM2.5 increased ROS generation, RAC1 activity, NOX1 expression, NLRP3 inflammasome (NLRP3, ASC, and caspase-1) activity, and IL-1β and IL-18 levels in cells; treatment with 10 μg/mL 8-OHdG significantly attenuated these effects. Furthermore, similar results, such as reduced expression of NOX1, NLRP3, ASC, and caspase-1, were observed in PM2.5-treated BEAS-2B cells when treated with an RAC1 inhibitor. These results show that 8-OHdG mitigates ROS generation and NLRP3 inflammation by inhibiting RAC1 activity and NOX1 expression in respiratory cells exposed to PM2.5.


Introduction
Particulate matter 2.5 (PM 2.5 ) has a small diameter (<2.5 µm) and large surface area [1,2]. Due to these characteristics, PM 2.5 has increased stagnation time and propagation distance, which causes it to easily infiltrate terminal airways such as the alveoli and readily dissolve into the circulatory system from the respiratory tract [1,2]. Since PM 2.5 also acts as a carrier of various materials such as nitrate, carbon particles, ammonium salt, bacteria, and viruses [3], PM 2.5 induces the secretion of various inflammatory cytokines

Cell Culture and Treatment
Human bronchial epithelial cells (BEAS-2B) were obtained from the ATCC (Manassas, VA, USA). The cells were maintained in DK-SFM medium (GIBCO BRL, Gaithersburg, MD, USA) supplemented with 1% keratin (GIBCO BRL) and 1% penicillin-streptomycin solution (Saturius, Kibbutz Beit-Haemek, Israel) and incubated in a humidified atmosphere containing 5% CO 2 . PM 2.5 was dissolved in 10% dimethyl sulfoxide, while 8-OHdG was dissolved in phosphate-buffered saline (PBS). For experiments involving PM 2.5 exposure, 5 or 10 µg/mL 8-OHdG was used. The growing cells were supplemented with serum-free culture medium and continuously incubated at 37 • C for 24 h. The PM 2.5 suspensions were always sonicated and vortexed before experiments.

Cell Viability Assay
Cell viability was measured using a Cell Counting Kit-8 (CCK-8 assay; DoJinDo Laboratories Co., Ltd., Kumamoto, Japan) and analyzed according to the manufacturer's instructions. Cells were seeded in a 96-well microplate at a density of 1 × 10 4 cells/well and treated with media containing various doses of PM 2.5 and 8-OHdG at 37 • C for 24 h. The absorbance of the samples was determined at 450 nm using a microplate reader (SpectraMAX ® i3X, Molecular Devices, San Jose, CA, USA). The experiment was performed in triplicate.

Cell Toxicity Assay
BEAS-2B cells were seeded into a 96 well-plate (1 × 10 4 cells/well) and exposed to PM 2.5 or 8-OHdG depending on the treatment group. Damaged cells were quantified by measuring the amount of LDH released into the culture medium.

Intracellular ROS Production
CellRox ® oxidative stress reagents (Sigma-Aldrich) were used according to the manufacturer's instructions to measure the intracellular ROS levels. BEAS-2B cells were seeded at a density of 2 × 10 4 cells/well in an eight-well chamber slide and incubated for 24 h, followed by treatment with PM 2.5 or 8-OHdG for another 24 h. After washing twice with PBS, 5 µM CellROX ® green reagent was added to the cells, which were incubated for 30 min at 37 • C away from light. DAPI was used to stain the nuclei. Next, the cells were incubated in 3.7% paraformaldehyde for 15 min at 37 • C to perform cell fixation. Observations were made using an inverted microscope (Flowview FV1000, Olympus, Tokyo, Japan), and intracellular fluorescence values (excitation = 485 nm; emission = 520 nm) were measured using Image J software (version 1.53t, 24 August 2022 (upgrade)).

Western Blotting Analysis
The cells were treated with or without PM 2.5 and 8-OHdG for 24 h. After centrifugation at 12,000 rpm for 10 min, 10 µg of total protein from each group was subjected to electrophoresis on SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore). After incubation in a blocking buffer (5% skim milk in 1× PBS containing 0.1% Tween-20) for 1 h at 37 • C, the membranes were incubated overnight with the primary antibodies at 4 • C. The membranes were then rinsed with PBS-T and probed with the corresponding secondary antibodies for 1 h at 37 • C. The blots were visualized using an Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore). Images of the Western blotting products were captured and analyzed using a ChemiDocTM Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Band density was measured using Image J software (version 1.53t, 24 August 2022 (upgrade)).

Detection of Active RAC1
An RAC1 pulldown assay was performed using a commercial kit (Cat No. 17-283, Millipore) according to the manufacturer's instructions. Briefly, cells were washed with icecold PBS and lysed with lysis buffer containing freshly added 1× protease inhibitor cocktail (ZenDEPOT, TX, USA) on ice for 5 min. After centrifugation at 14,000× g for 15 min at 4 • C, the supernatant was mixed with glutathione agarose (Sigma-Aldrich) resins and incubated at 4 • C for 1 h. The resin was then washed three times with 1× cell lysis/binding/wash buffer. The resin-bound GTP-RAC1 was eluted with a 5× SDS sample buffer, followed by Western blotting analysis using mouse anti-human RAC1 antibodies.

ELISA
BEAS-2B cells seeded in six-well plates were separately treated with 10 µg/mL PM 2.5 or 8-OHdG 5 for 24 h. The culture medium was then collected and stored at −80 • C until they were assayed for IL-1β, IL-6, and IL-18 using ELISA kits according to the manufacturer's instructions. To further explore whether 8-OHdG decreases PM 2.5 -induced inflammatory cytokine levels, we concurrently exposed the cells to 80 µg/mL PM 2.5 and 5 or 10 µg/mL 8-OHdG, and estimated the IL-1β, IL-6, and IL-8 levels in the medium. The absorbance was determined at 450 nm (excitation) and at 570 nm (emission) using a microplate reader (SpectraMAX ® i3X, Molecular Devices, San Jose, CA, USA).

Statistical Analysis
Student's t-test was used to analyze the data. All statistical analyses were performed using SPSS software (SPSS 10.0 version, New York, NY, USA). Data are presented as the mean ± standard deviation. p < 0.05 was considered a statistically significant difference.

PM 2.5 Exposure Decreased BEAS-2B Cell Viability
The appropriate concentrations for the PM 2.5 and 8-OHdG treatments were determined using the CCK-8 or lactate dehydrogenase (LDH) assays. BEAS-2B cell viability significantly decreased at PM 2.5 concentrations higher than 40 µg/mL in the PM 2.5 -treated group ( Figure 1A). The cell viability decreased by 20% and 38% in the groups treated with 80 µg/mL and 160 µg/mL PM 2.5 , respectively (n = 3, p < 0.001 vs. the control and vehicle groups). However, no significant difference in cell viability was observed between the control and treatment groups at 8-OHdG concentrations of 1-200 µg/mL ( Figure 1B).
On the basis of these cell viability and cytotoxicity results, 80 µg/mL was chosen as the PM 2.5 concentration for further in vitro experiments because this concentration elicited an inflammatory response and induced 20-30% cytotoxicity.   CCK and LDH assays were performed to evaluate the effect of 8-OHdG treatment on cell viability and toxicity in PM 2.5 -treated BEAS-2B cells. As shown in Figure 1C,D, cell viability decreased to 28% after treatment with 80 µg/mL PM 2.5 and was notably recovered by 15% following 8-OHdG treatment. Similarly, PM 2.5 -induced cytotoxicity was notably inhibited by 46% following treatment with 8-OHdG ( Figure 1D). Furthermore, the attenuating effect of 8-OHdG was higher at 10 µg/mL than at 5 µg/mL. Hence, further experiments were performed to evaluate the efficacy of 8-OHdG in decreasing NLRP3 inflammasome activation and pyroptosis induction via RAC1 inhibition at a concentration of 10 µg/mL.

Exogenous 8-OHdG Treatment Decreased ROS Production in PM 2.5 -Treated BEAS-2B Cells
Intracellular ROS levels were measured using a CellRox ® oxidative stress assay to determine whether 8-OHdG reduced PM 2.5 -induced ROS production. BEAS-2B cells exposed to PM 2.5 displayed brighter green fluorescence than the control. The cells simultaneously pretreated with 8-OHdG and PM 2.5 displayed light-green fluorescence similar to the control ( Figure 1E). We quantified the ROS levels by measuring the fluorescence intensity and confirmed that intracellular ROS levels were elevated in BEAS-2B cells exposed to 80 µg/mL PM 2.5 but significantly decreased after incubation with 8-OHdG ( Figure 1F).

Exogenous 8-OHdG Treatment Decreased ROS Production in PM2.5-Treated BEAS-2B Cells
Intracellular ROS levels were measured using a CellRox ® oxidative stress assay to determine whether 8-OHdG reduced PM2.5-induced ROS production. BEAS-2B cells exposed to PM2.5 displayed brighter green fluorescence than the control. The cells simultaneously pretreated with 8-OHdG and PM2.5 displayed light-green fluorescence similar to the control ( Figure 1E). We quantified the ROS levels by measuring the fluorescence intensity and confirmed that intracellular ROS levels were elevated in BEAS-2B cells exposed to 80 µg/mL PM2.5 but significantly decreased after incubation with 8-OHdG (Figure 1F).

Exogenous 8-OHdG Attenuated PM2.5-Induced Inflammatory Cytokine Secretion
To quantify the levels of inflammatory cytokines released by BEAS-2B cells, we analyzed IL-1β, IL-6, and IL-8 expression using enzyme-linked immunosorbent assay (ELISA) kits. As expected, PM2.5 treatment showed significantly increased expression levels of the inflammatory cytokines IL-1β, IL-6, and IL-8 in a concentration-dependent manner compared with the control and vehicle-treated groups ( Figure 2).
Simultaneous exposure of the cells to PM2.5 and 8-OHdG showed that pretreatment significantly reduced IL-1β, IL-6, and IL-18 expression (Figure 2). These results prove that 8-OHdG pretreatment recovered cell viability, mitigated cytotoxicity, and reduced ROS production and PM2.5-induced inflammatory cytokine secretion.  Simultaneous exposure of the cells to PM 2.5 and 8-OHdG showed that pretreatment significantly reduced IL-1β, IL-6, and IL-18 expression (Figure 2). These results prove that 8-OHdG pretreatment recovered cell viability, mitigated cytotoxicity, and reduced ROS production and PM 2.5 -induced inflammatory cytokine secretion.

Exogenous 8-OHdG Alleviated NLRP3 Inflammasome Activation in PM 2.5 -Treated BEAS-2B Cells
To further assess the role of PM 2.5 in epithelial cell inflammation, we performed Western blotting to detect NLRP3 inflammasome activation. The expression of NLRP3, ASC, cleaved caspase-1, precursor IL-1β, and mature IL-1β significantly increased in PM 2.5treated BEAS-2B cells compared with that in the control group ( Figure 3A,B). However, the expression of inflammasome-related proteins significantly decreased after preincubation with 8-OHdG. These results indicated that 8-OHdG attenuates the NLRP3/caspase-1 pathway activated by PM 2.5 treatment.

Exogenous 8-OHdG Inhibits NOX1 Expression and RAC1 Activation in PM 2.5 -Treated BEAS-2B Cells
Previous studies state that exogenous 8-OHdG alleviates liver fibrosis and epithelialmesenchymal transition by inhibiting NOX-derived ROS formation [34]. Therefore, we determined the protein expression of NOX1, NOX2, NOX3, and NOX4 using immunoblotting and found that only NOX1 expression was significantly elevated in BEAS-2B cells exposed to PM 2.5 compared with that of the control group. However, pretreatment with 8-OHdG inhibited NOX1 expression in PM 2.5 -treated cells ( Figure 4A).
The NADPH oxidase NOX1 consists of the transmembrane proteins NOX1 and p22 phox and the cytosolic proteins NOXO1, NOXA1, and RAC1 [24]. Here, we additionally investigated p22 phox expression and RAC1 activation. Figure 4B,C show that the PM 2.5induced increase in p22 phox expression and RAC1 activation were significantly reversed after treatment with 8-OHdG. These results show that 8-OHdG reduces ROS generation by modulating NOX1/RAC1 activation.
The NADPH oxidase NOX1 consists of the transmembrane proteins NOX1 and p22 phox and the cytosolic proteins NOXO1, NOXA1, and RAC1 [24]. Here, we additionally investigated p22 phox expression and RAC1 activation. Figure 4B,C show that the PM2.5-induced increase in p22 phox expression and RAC1 activation were significantly reversed after treatment with 8-OHdG. These results show that 8-OHdG reduces ROS generation by modulating NOX1/RAC1 activation.

RAC1 Inhibitor Downregulates NOX1/p22 phox Expression in PM2.5-Treated BEAS-2B Cells
NSC23766 is a specific RAC1 inhibitor that can be used to confirm the effect of 8-OHdG on RAC1-mediated NOX1/p22 phox expression and NLRP3 signaling. After treatment for 24 h with the RAC1 inhibitor, cell viability was measured using the CCK assay. NSC23766 had no effect on cell viability at concentrations of 0.1-10 µM ( Figure S1). We preincubated the cells with NSC23766 and 8-OHdG and subsequently exposed them to PM2.5.

RAC1 Inhibitor Downregulates NOX1/p22 phox Expression in PM 2.5 -Treated BEAS-2B Cells
NSC23766 is a specific RAC1 inhibitor that can be used to confirm the effect of 8-OHdG on RAC1-mediated NOX1/p22 phox expression and NLRP3 signaling. After treatment for 24 h with the RAC1 inhibitor, cell viability was measured using the CCK assay. NSC23766 had no effect on cell viability at concentrations of 0.1-10 µM ( Figure S1). We preincubated the cells with NSC23766 and 8-OHdG and subsequently exposed them to PM 2.5 .

RAC1 Inhibitor Downregulates NOX1/p22 phox Expression in PM2.5-Treated BEAS-2B Cells
NSC23766 is a specific RAC1 inhibitor that can be used to confirm the effect of 8-OHdG on RAC1-mediated NOX1/p22 phox expression and NLRP3 signaling. After treatment for 24 h with the RAC1 inhibitor, cell viability was measured using the CCK assay. NSC23766 had no effect on cell viability at concentrations of 0.1-10 µM ( Figure S1). We preincubated the cells with NSC23766 and 8-OHdG and subsequently exposed them to PM2.5.

RAC1 Inhibitor Attenuates NLRP3 Inflammasome and Inflammatory Responses in PM 2.5 -Treated BEAS-2B Cells
Western blotting showed a significantly higher expression of NLRP3, ASC, cleaved caspase-1, precursor IL-1β, and mature IL-1β proteins in BEAS-2B cells treated with 80 µg/mL PM 2.5 than in the control and vehicle groups ( Figure 5C,D), similar to previous results. However, the expression of these proteins was reduced in PM 2.5 -treated cells after preincubation with an RAC1 inhibitor or 8-OHdG.
In our study, PM 2.5 treatment increased the expression of NLRP3, ASC, and cleaved caspase-1 and the levels of pro-IL-1β and mature IL-1β in BEAS-2B cells. Similarly, IL-1β and IL-18 levels were elevated in PM 2.5 -treated BEAS-2B cells. However, treatment with 8-OHdG attenuated these effects. Furthermore, the mRNA expression of NOX1, inflammation-related NLRP3 signaling molecules, and inflammatory cytokines was increased in BEAS-2B cells upon exposure to PM 2.5 . Conversely, their expression decreased after treatment with 8-OHdG (Supplementary Figure S2 and Supplementary Table S1).
The molecule 8-OHdG is known to exert inhibitory effects on RAC1 by binding with the RAC1-GEF complex [57]. This study demonstrated a reduction in the PM 2.5 -induced increase in RAC1 activity and NOX1 expression following treatment with 10 µg/mL 8-OHdG. Given that RAC1 activation is required to activate NOX1 and NOX2 [27], 8-OHdG seems to decrease NOX1 activity by inhibiting RAC1.
NSC23766 specifically prevents the conversion of RAC1-GDP to RAC1-GTP by competitively blocking the binding loop of RAC1-specific GEF [58]. Adding an RAC1 inhibitor to PM 2.5 -treated BEAS-2B cells decreased the expression of NOX1 and p22 phox . Interestingly, the degree to which 8-OHdG attenuated the expression of these proteins was similar to that observed for the RAC1 inhibitor. NLRP3 inflammasome activation, which was evaluated on the basis of NLRP3, ASC, and cleave caspase-1 expression, was also decreased by both 8-OhdG and the RAC1 inhibitor to similar degrees. The expression of pro-IL-1β and IL-1β in PM 2.5 -treated BEAS-2B cells was also decreased by both 8-OhdG and the RAC1 inhibitor.
IL-1β is a pluripotent proinflammatory cytokine involved in various inflammationrelated lung diseases [59]. Moreover, IL-1β leads to PM 2.5 -induced pulmonary inflammation [60]. IL-1β triggers leukocytes to secrete more cytokines, such as IL-6, and increases the migration of leukocytes, which enhances inflammation [61]. IL-1β is also involved in increasing IL-18 expression, which promotes the T-cell-induced immune response in airway inflammation [62,63]. IL-6 is an essential proinflammatory cytokine, the levels of which increase during acute inflammation due to environmental insult to the lungs [62,63].
In this study, we also observed increased IL-1β and IL-18 levels and IL-6 secretion in BEAS-2B cells following exposure to PM 2.5 , which decreased following treatment with 8-OHdG.
Reportedly, the average life span is shortened by 8.6 months by PM 2.5 [64], which constitutes approximately 70% of inhalable particles in humans [65]. Therefore, increased daily exposure to PM 2.5 by 10 µg/m 3 increases the incidence of respiratory diseases by 2.07% [66,67]. As PM 2.5 exerts a tremendous impact on the respiratory tract by increasing ROS concentration or inflammation that results in lung injury, various modalities to decrease ROS levels or inflammation are being widely evaluated. Exogenous 8-OHdG, which decreases ROS levels and attenuates inflammation by inhibiting RAC1 and NOXs, could be a viable candidate for decreasing PM 2.5 -induced lung injury. Nevertheless, this study had limitations. First, a broader spectrum of concentrations must be shown since 8-OHdG is known to exert divergent effects due to its oxidative potential. Our experiments were performed using only two doses, 5 and 10 µg/mL. We focused on verifying whether low concentrations of 8-OHdG reduce the oxidative stress induced by PM 2.5 . Thus, although treatment with 8-OHdG alone did not affect cell viability, it was difficult to observe its effect on other categories of markers, such as inflammation. Further studies will be needed to explain the effect of different concentrations of 8-OHdG on these parameters.
Second, although the ATCC recommended that BEAS-2B cells be cultured in BEGM, other media could be used for BEAS-2B culture. According to Zhao and Klimecki, fetal bovine serum showed cytotoxicity in BEAS-2B cells and decreased the expression of E-cadherin, an epithelial cell phenotype marker [68]. When cells lose their original characteristics, they exhibit various responses to stimuli, usually at varying degrees. We were not able to further examine the difference between the culture conditions and PM 2.5 concentrations in BEAS-2B cells, which could have provided more information to better understand the degree of NOX expression after PM 2.5 exposure. Therefore, it is necessary to characterize the cells more comprehensively according to the composition of the culture medium.
Research to address these limitations will continue in the future. In this study, we found that exogenous 8-OHdG, which reduces ROS levels and attenuates inflammation by inhibiting RAC1 and NOX, can reduce lung damage caused by PM 2.5 exposure and suggested possible candidates.

Conclusions
Our study showed that exogenous 8-OHdG decreased PM 2.5 -induced ROS generation and NLRP3 inflammasome formation, which was accompanied by reduced IL-6, IL-1β, and IL-18 levels in respiratory cells. The possible mechanism underlying these effects of 8-OHdG could be the inhibition of RAC1 and NOX1.

Conflicts of Interest:
The authors declare no conflict of interest.