ROS Generated by Upconversion Nanoparticle-Mediated Photodynamic Therapy Induces Autophagy via PI3K/AKT/Mtor Signaling Pathway in M1 Peritoneal Macrophage
Xiaobo Hana Jiayuan Koub Yinghong Zhenga Zhongni Liua Yueqing Jianga Ziyu Gaoa Lin Conga Liming Yanga
aDepartment of Pathophysiology, Key Laboratory of Cardiovascular Pathophysiology, Harbin Medical University, Harbin, China, bDepartment of Biochemistry and Molecular Biology, Harbin Medical University, Harbin, China
Key Words
Reactive oxygen species Autophagy M1 Peritoneal macrophage Upconversion nanoparticle Photodynamic therapy
Abstract
Background/Aims: Atherosclerosis is a chronic inflammatory cardiovascular disease. Macrophages are major components of atherosclerotic plaques and play a key role in the development of atherosclerosis by secreting a variety of pro-inflammatory factors. Our previous studies have confirmed that upconversion nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) mediated photodynamic therapy (PDT) can promote cholesterol efflux and induce apoptosis in THP-1 macrophages. In this study, we investigated whether reactive oxygen species (ROS) generated by UCNPs-Ce6-mediated PDT can induce autophagy to inhibit the expression of pro-inflammatory factor in M1 peritoneal macrophages. Methods: Peritoneal macrophages were collected from C57/BL6 mice injected with 3% thioglycollate broth medium and induced by lipopolysaccharides and interferon-γ. Intracellular ROS production was assessed by 2′-7′-dichloroflorescein diacetate and flow cytometry. Autophagy was assayed by western blot, transmission electron microscopy and immunofluorescence. Pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay and western blot. Results: Model M1 peritoneal macrophages were established after 24 h induction. Protein expression levels of LC3 II and Beclin1, and degradation of p62 increased and peaked at 2 h in the PDT group. Meanwhile, levels of inflammatory cytokines iNOS, IL-12, and TNF-α markedly decreased after PDT. The increase in autophagy levels and decrease in pro-inflammatory cytokines were significantly inhibited by 3-methyladenine. Furthermore, ROS generated by UCNPs-Ce6 mediated PDT activated autophagy. The expression of autophagy related-protein and inflammatory cytokines iNOS, IL-12, and TNF-α were inhibited by the ROS inhibitor N-acetyl cysteine. Conclusion: ROS generated by UCNPs-Ce6-mediated PDT activated autophagy and inhibited the expression of pro-inflammatory factors of M1 peritoneal macrophage via the PI3K/AKT/mTOR signaling pathway.
Introduction
Atherosclerosis is a common cardiovascular disease, and a serious threat to health, and it is generally believed that inflammation is the main cause. Macrophages are involved in the progression of the atherosclerosis [1-3]. Macrophages are mainly divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, and their proportion in atherosclerosis plaque changes dynamically with the development of the disease. M1 macrophages secrete pro-inflammatory factors causing unstable plaques, which eventually rupture and cause clinical symptoms [4, 5]. Therefore, by inhibiting the expression of pro-inflammatory factors secreted by M1 macrophages, plaque inflammation can be reduced and plaque stability increased [5, 6].
Autophagy is involved in the metabolism of proteins, glucose, and lipids, and is central to intracellular homeostasis and development [7, 8]. Furthermore, autophagy dysfunction is closely associated with cardiovascular disease [9, 10]. Numerous studies have demonstrated that autophagy plays an important anti-inflammatory role in atherosclerosis [11-13]. Deficient macrophage autophagy increases plaque instability by activating inflammation, which suggests that intact autophagy processes are essential for reducing the occurrence of adverse vascular events through the suppression of inflammation [14, 15].
Photodynamic therapy (PDT) is a new therapeutic strategy for cardiovascular disease and is non-invasive, accurate, and has few side effects. PDT mainly involves three important components: the photosensitizer, light and oxygen molecules [16-19]. The photosensitizer is a vital component of PDT. Accordingly, we chose a composite photosensitizer UCNPs-Ce6 as a medium, which can be used with reasonable efficiency together with a 980 nm laser [20, 21]. In contrast to Photofrin, 5-aminolevulinic acid, and even third-generation photosensitizers, which are activated by visible light (660 nm), the UCNP-Ce6 photosensitizer is activated by near-infrared (NIR) light (980 nm). The penetration depth of NIR light (980 nm) is deeper than that of the visible light (660 nm). UCNPs-Ce6 increases the penetration depth of laser during PDT [22, 23]. Our prior studies have confirmed that PDT can promote cholesterol efflux by inducing autophagy and induce THP-1 macrophage apoptosis to attenuate the progression of atherosclerosis [24-26]. This provides an important basis for UCNPs-Ce6 mediated PDT to inhibit the expression of inflammatory factor in this study.
Here, we investigated whether UCNPs-Ce6-PDT could induce autophagy to inhibit inflammation by the generation of reactive oxygen species (ROS) via the PI3K/AKT/mTOR signaling pathway in M1 peritoneal macrophages.
Materials and Methods
980-nm PDT device
The 980-nm PDT device was developed by the Harbin Institute of Technology (Harbin, China). The laser generator emits single 980-nm wavelength irradiation. For PDT treatment, the distance from the cell to the laser generator is 2 cm with a laser irradiation density of 1.5 W/cm2 [27, 28].
PDT treatment protocols
UCNPs-Ce6 was designed by the Harbin Institute of Technology. Approximately 1 mg/mL of stock solution in dimethyl sulfoxide was stored at 4 °C in the dark.
For the inhibition studies, 1 mM of the ROS inhibitor N-acetyl cysteine (NAC; Sigma-Aldrich, St. Louis, MO, USA), 10 mM of the autophagy inhibitor 3-methyladenine (3-MA; Sigma-Aldrich), 10 μM of the PI3K inhibitor LY294002 (Sigma-Aldrich) and 20 mM of the AKT inhibitor triciribine (Sigma-Aldrich) were incubated together with UCNPs-Ce6 60 min before UCNPs-Ce6-mediated PDT.
Cell culture
Peritoneal macrophages were collected from C57/ BL 6 mice injected with 2 mL of 3% thioglycollate broth medium for 72 h. The cells were cultured in RPMI 1640 medium (HyClone, Laboratories Inc., South Logan, UT, USA) containing 10% fetal bovine serum (FBS; HyClone, Laboratories Inc), and 20 μg/mL of penicillin and streptomycin (Sigma-Aldrich, USA) for 24 h. Cells were maintained at 37 °C in a humidified incubator with 5% CO2. For the experiments, the cells were seeded in 35-mm Petri dishes or 96-well plates at a density of 1.0 × 105 cells/mL. For M1 induction, 100 ng/mL of lipopolysaccharide (LPS; Sigma-Aldrich) and 20 ng/mL of interferon-γ (IFN-γ; PeproTech, Rocky Hill, NJ, USA) were added into the culture medium for 24 h. Cells were then collected, differentiated and randomly divided into four groups: (1) control, (2) laser alone, (3) UCNPs-Ce6 alone, (4) PDT.
Cell viability assay
The cell survival rate was detected by the Cell Counting Kit 8 (CCK-8) assay (Beyotime Biotechnology, Jiangsu, China). M1 peritoneal macrophages were seeded in 96-well culture plates. The plate was carefully washed with phosphate-buffered saline (PBS) and 100 μL medium containing CCK-8 (RPMI 1640 medium and CCK-8 volume ratio of 9:1) was added into the wells after the different treatments. After incubation for 30 min at 37 °C in the dark, the absorption of each well was measured by a microplate reader (Varian Australia Pty Ltd, Melbourne, Australia) at 450 nm. All experiments were repeated three times.
Detection of intracellular ROS
Intracellular ROS were analyzed by 2′-7′-dichloroflorescein diacetate (DCFH-DA; Beyotime Biotechnology). After PDT, the cells were washed with PBS and incubated with 20 μM DCFH-DA diluted in RPMI 1640 medium for 30 min at 37 °C in the dark. Cells were then washed with PBS, and then measured under a fluorescence microscope (Olympus IX81, Olympus, Tokyo, Japan) at 488 nm excitation and 525 nm emission wavelengths. For the flow cytometry assay, cells were analyzed using a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). All experiments were repeated three times.
Transmission electron microscopy
Following different treatments, the cells were harvested by centrifugation and fixed with 2.5% glutaraldehyde at 4 °C overnight. Cells were then measured by transmission electron microscopy (JEM-1220, JEOL, Tokyo, Japan). All experiments were repeated three times.
Fluorescence staining
After the treatments, cells were washed with PBS, and incubated with 500 μg/mL CD16/32 and tumor necrosis factor (TNF-α; Affymetrix eBioscience, USA) for 30 min at 37 °C in the dark. After washing with PBS, the cells were observed under a fluorescence microscope (Olympus IX81). All experiments were repeated three times.
Immunofluorescence staining
At the indicated times after PDT, cells were washed with PBS, and fixed with 4% paraformaldehyde for 30 min and permeabilized with 1% Triton X-100 for 20 min at room temperature. Cells were washed with PBS, blocked with 3% bovine serum albumin, and incubated with Lamp2 and LC3 antibodies (1:200) overnight at 4 °C. Cells were again washed with PBS and incubated with the corresponding secondary antibody at 37 °C temperature for 1 h in the dark. After washing with PBS, the cells were observed by laser scanning confocal microscope (LSCM; Meta, Germany). All experiments were repeated three times.
Monodansylcadaverine staining
Monodansylcadaverine (MDC; Sigma-Aldrich) was used to observe the autophagic vacuole. After the different treatments, cells were incubated with 50 μmol/mL MDC for 30 min at 37 °C in the dark. After washing with PBS, the cells were observed under a fluorescence microscope (Olympus IX81). All experiments were repeated three times.
Acridine orange staining
Acridine orange staining (Sigma-Aldrich) was used to observe acidic vesicle organelles. At 2 h after the different treatments, the cells were incubated with 0.01% acridine orange for 30 min at 37 °C in the dark. After washing twice with PBS, the cells were observed under a fluorescence microscope (Olympus IX81). All experiments were repeated three times.
Western blot analysis
After the treatments, RIPA lysis buffer was used to extract the total protein. Protein concentrations were assessed via a bicinchoninic acid kit (Beyotime Biotechnology). After quantification and denaturation, the protein samples were electrophoresed in sodium dodecyl sulfate-polyacrylamide gel and transferred onto a 0.45 μm polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). Blocking was achieved with 5% low-fat milk diluted with Tris-buffered saline and Tween 20, and the membranes were incubated with primary antibodies at 4 °C overnight. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP) - conjugated secondary antibodies for 2 h at room temperature. After a further wash with TBST, the immune complexes were detected using an enhanced chemiluminescence reagent. The protein bands were quantified using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). All experiments were repeated three times.
Antibodies against Beclin1, p62, LC3B, mTOR, p-mTOR, AKT, and p-AKT (1:1000) were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against iNOS, IL-12 and TNF-α (1:1000) were purchased from Abcam (Burlingame, CA, USA). Antibodies against β-actin (1:1000) were purchased from Proteintech Group (Wuhan, China). The HRP-conjugated secondary mouse and rabbit antibodies (1:5000) were purchased from ZhongShan Company (Beijing, China).
Enzyme linked immunosorbent assay
To detect cytokines released by M1 peritoneal macrophages, cell supernatants were collected after the different treatments, and the cytokines of iNOS, IL-12 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA) kits (Elabscience Biotechnology Co, Ltd, Hubei, China). All experiments were repeated three times.
Statistical analysis
All experiments were repeated at least three times independently. The data were analyzed using one-way analysis of variance and the independent test. The results are presented as the mean ħ standard deviation. P<0.05 was considered statistically significant.
Results
Establishment of M1 peritoneal macrophage model
To establish the M1 peritoneal macrophage model, LPS and IFN-γ were added into the culture medium with peritoneal macrophages. After induction for 24 h, protein expression and the secretion of iNOS, IL-12, and TNF-α in the supernatant increased significantly (Fig. 1A, D). Subsequently, we verified the presence of specific fluorescence markers CD16/32, and TNF-α and pro-inflammatory factor proteins of M1 peritoneal macrophages, the expression of green fluorescence and protein level increased significantly (Fig. 1B, C). All results further confirmed the establishment of the M1 peritoneal macrophage model.
Optimization of UCNPs-Ce6-mediated PDT conditions for M1 peritoneal macrophages
To choose the optimal PDT time, intensity, and photosensitizer dose, cell viability was determined with the CCK-8 assay after different treatments. By screening the different concentrations and laser intensities, a dose of 6 μg/mL of UCNPs-Ce6 and a laser intensity of 1.5 W/cm2 were considered safe and had no significant effect on cell viability (Fig. 2A, B). Furthermore, we tested a combination of drug concentration and time (Fig. 2C), laser intensity and time (Fig. 2D), and drug concentration and laser intensity (Fig. 2E) via CCK-8 assay. Accordingly, 6 μg/mL of UCNPs-Ce6 and laser irradiation at 1.5 W/cm2 of 60 s were selected as the optimal conditions for the M1 peritoneal macrophages (Fig. 2F).
UCNPs-Ce6-mediated PDT inhibited the expression and secretion of pro-inflammatory factors in M1 peritoneal macrophages
To investigate the effects of PDT on the pro-inflammatory factors of M1 peritoneal macrophages，the expression of pro-inflammatory factor proteins was tested at different induction times. As shown in Fig. 3A, the inflammatory factors decreased significantly at 12 h after PDT. Pretreatment with 3-MA increased the expression and secretion of pro-inflammatory factors (Fig. 3B, C). Moreover, compared with the other groups, the secretion of the pro-inflammatory factors in the supernatants of the PDT group was also significantly decreased at 12 h after PDT (Fig. 3D). Thus, PDT inhibited the expression of pro-inflammatory factors in M1 peritoneal macrophages. Next，we verified the specific fluorescence markers CD16/32 and TNF-α of M1 peritoneal macrophages. The intensity of green fluorescence also decreased significantly in the PDT group, and was inhibited by 3-MA (Fig. S1A, B - for all supplemental material see www.cellphysiolbiochem.com). These results suggested that PDT inhibited the expression and secretion of pro-inflammatory factors in M1 peritoneal macrophages through the activation of autophagy.
UCNPs-Ce6-mediated PDT induced autophagy in M1 peritoneal macrophage
To verify whether the UCNPs-Ce6-mediated PDT could induce autophagy in M1 peritoneal macrophages, autophagy related-proteins were examined at different time points. We found that UCNPs-Ce6-mediated PDT upregulated LC3 II and Beclin1 expression and downregulated p62 expression at 2 h (Fig. 4A). Subsequently, we used the autophagy broad-spectrum inhibitor 3-MA and found that LC3 II conversion, Beclin1 expression and p62 degradation were suppressed by 3-MA after PDT (Fig. 4B). In addition, autophagosome ultrastructures of M1 peritoneal macrophages were observed by transmission electron microscopy after the different treatments. Compared with the other groups, macrophages in the PDT group appeared to be typical autophagosomes with phospholipid bilayers, and this formation was inhibited by 3-MA (Fig. 4C). Moreover, the co-localization of Lamp2 and LC3 detected after PDT further confirmed autophagy (Fig. 4D). Additionally, we investigated autophagic flux after PDT with MDC and acridine orange staining. The results indicated that the green fluorescence in the PDT group was brighter and the red fluorescence was more obvious than in macrophages of the other groups (Fig. 4E, F). Therefore, UCNPs-Ce6-mediated PDT induced autophagy in M1 peritoneal macrophages.
UCNPs-Ce6-mediated PDT induced autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway in M1 peritoneal macrophages
The PI3K/AKT/mTOR signaling pathway is the classic pathway of autophagy [29]. As shown in Fig. 5A, the expression of p-AKT and p-mTOR had clearly decreased at 2 h after PDT. Additionally, changes in the expression of p-AKT and p-mTOR were not obvious, but the levels of autophagy associated protein LC3 II significantly increased after pretreatment with PI3K inhibitor LY294002 and AKT inhibitor triciribine (Fig. 5B, C). These results indicated that UCNPs-Ce6-PDT induced autophagy through the PI3K/AKT/mTOR signaling pathway.
UCNPs-Ce6-mediated PDT promoted ROS generation in M1 peritoneal macrophages and induced autophagy by suppressing the PI3K/AKT/ mTOR signaling pathway
The generation of ROS represents the foundation of PDT [30]. Compared with other groups, significant green fluorescence was observed in the PDT group with DCFH-DA staining. The increase in ROS production was inhibited by pretreatment with NAC (Fig. 6A). Furthermore, the generation of ROS was examined by flow cytometry, and ROS levels increased significantly after UCNPs-Ce6-PDT. ROS production was effectively attenuated by NAC (Fig. 6B).
Next, we investigated the effect of ROS on PDT induced autophagy. The levels of expression of autophagy-related proteins and PI3K/AKT/mTOR signaling pathway related proteins were examined by pretreatment with NAC. The results showed that the expression of Beclin1, the transformation of LC3 II and the degradation of p62 were inhibited by NAC. Simultaneously, the expression of p-mTOR and p-AKT clearly increased compared with the PDT group (Fig. 6C, D). To further validate the relationship between autophagy and ROS, acridine orange and MDC staining was used to observe the acidic vesicular organelles. Increased green and red fluorescence was found in the PDT group, and attenuated by NAC (Fig. S2A, B). Moreover, the co-localization of Lamp2 and LC3 induced by PDT was inhibited by NAC (Fig. S2C).
UCNPs-Ce6-mediated PDT inhibited the expression and secretion of pro-inflammatory factors through the regulation of ROS in M1 peritoneal macrophages
To investigate the relationship between ROS and pro-inflammatory cytokines, the protein expression levels of iNOS, IL-12, and TNF-α and the pro-inflammatory factors in the supernatants were examined by pretreatment with NAC. The results indicated that, compared with the PDT alone group, the expression and secretion of pro-inflammatory factors clearly increased with NAC pretreatment (Fig. 7A, B). We then examined the specific fluorescence markers CD16/32 and TNF-α of M1 peritoneal macrophages and found that the intensity of green fluorescence markers also increased significantly in the PDT-NAC group (Fig. 7C). These experiments indicated that PDT inhibited the expression and secretion of pro-inflammatory factors through the regulation of ROS in M1 peritoneal macrophages.
Discussion
In this study, ROS generated by UCNPs-Ce6-mediated PDT inhibited the expression of pro-inflammatory factors by inducing autophagy in M1 peritoneal macrophages. In addition, the PI3K/AKT/mTOR signaling pathway was involved in PDT-activated autophagy. Thus, our findings revealed an anti-inflammatory effect through autophagy induced by PDT, suggesting that UCNPs-Ce6-mediated PDT is a potential treatment for atherosclerosis.
ROS are crucial mediators of inflammation. It has been found that ROS can induce cell apoptosis and autophagy by regulating multiple signaling pathways, which plays an important role in various diseases [31-33]. Similarly, ROS, as products of PDT, have important biological effects in the treatment of cardiovascular disease [2, 5]. In the present study, a significant increase in ROS generation was detected by flow cytometry and DCFH-DA in the PDT group, demonstrating that PDT was capable of producing ROS, which was clearly inhibited by NAC. Furthermore, our results indicated that the generation of PDT-induced ROS could activate autophagy in M1 peritoneal macrophages, which was also restrained by NAC. It was reported that ATG4 was identified as an effector molecule of ROS to promote LC3 lipogenesis, which is a key step of autophagy initiation [34]. Additional studies should be performed to verify it. In addition, previous reports have shown that ROS can not only inhibit but also promote the expression of pro-inflammatory cytokines through multiple signaling pathways [35-37]. In our experiments, ROS induced by PDT clearly inhibited the expression of pro-inflammatory factors. Interestingly, our previous studies verified that ROS generated by 1.0 W/cm2 and 8 μg/mL UCNPs-Ce6-PDT induced autophagy in THP-1 macrophage-derived foam cells and 2.0 W/cm2 and 16 μg/mL UCNPs-Ce6-PDT induced apoptosis in THP-1 macrophages [5, 24]. In the present study, we verified that ROS generated by 1.5 W/cm2 and 6 μg/mL UCNPs-Ce6-PDT induced autophagy in M1 peritoneal macrophages. Therefore, distinct degrees of ROS produced by different intensities of laser may play diverse roles in the treatment of atherosclerosis.
Autophagy is a complex physiological process, and is indispensable for the reconstruction, regeneration, and repair of cells, as well as for the maintenance of cellular and organismal homeostasis [38-40]. Autophagy also play an important role in cardiovascular system, with autophagy dysfunction-contributing to the development of cardiovascular diseases such as atherosclerosis [41-43]. In this study, we found that PDT induced autophagy in a time-dependent manner, autophagy peaked at 2 h, and these results were blocked by 3-MA. Moreover, typical autophagosomes with a phospholipid bilayer was found in the PDT group. MDC and acridine orange staining as well as the co-localization of LC3 and Lamp2 confirmed that autophagy was induced by PDT. Next, previous studies have confirmed that autophagy can promote cholesterol efflux, reduce lipid accumulation and inhibit the secretion of inflammatory factors in THP-1 macrophages [5, 36, 44]. In our study, PDT also inhibited the expression and secretion of pro-inflammatory factors in M1 peritoneal macrophage, and these effects were inhibited by 3-MA. Our results show that PDT inhibited the expression of pro-inflammatory factors by inducing autophagy in M1 peritoneal macrophage, which is consistent with studies of autophagy for the suppression of inflammation [45, 46]. It was reported that Toll-like receptor 4 (TLR4) was believed as a potential target to govern the pro-inflammatory reaction through autophagy [47]. Future study should be performed to better understand how autophagy induced by PDT inhibit the expression of pro-inflammatory factors.
Inhibition of the PI3K/AKT/mTOR signaling pathway could induce autophagy and enhance the expression of autophagy-related proteins [48-50]. In the present study, p-AKT, p-mTOR, and LC3II/LC3I were detected by pretreatment with the PI3K inhibitor LY294002 and AKT inhibitor triciribine, p-AKT and p-mTOR were not affected by LY294002 and triciribine, while LC3 II/LC3 I conversion clearly increased. Furthermore, the expression of p-AKT and p-mTOR increased with NAC pretreatment. These results demonstrated that autophagy was induced by PDT through the PI3K/AKT/mTOR signaling pathway via ROS generation in M1 peritoneal macrophages.
As illustrated in Fig 8, these experiments confirmed that the UCNPs-Ce6-PDT enhanced the generation of ROS in M1 peritoneal macrophages, and the ROS induced autophagy through suppressing PI3K/AKT/mTOR signaling pathway. Furthermore, autophagy induced by ROS inhibited the expression of pro-inflammatory factors in M1 peritoneal macrophages.
Conclusion
ROS generated by UCNPs-Ce6-mediated PDT inhibited the expression of pro-inflammatory factors by activating autophagy via the PI3K/AKT/mTOR signaling pathway in M1 peritoneal macrophages.
Acknowledgements
This study was supported by the Foundation of Key Laboratories of Myocardial Ischemia Mechanism and Treatment (Harbin Medical University) (KF201814) and the Wu Liande Youth Science Foundation of Harbin Medical University (WLD-QN1104).
Disclosure Statement
The authors declare that there are no conflicts of interest.
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