Cigarette smoke extract-induced inflammatory response via inhibition of the TFEB-mediated autophagy in NR8383 cells

Abstract

Objective: Chronic pulmonary inflammation caused by long-term smoking is the core pathology of COPD. Alveolar macrophages (AMs) are involved in the pulmonary inflammation of COPD. The accumulation of damaged materials caused by impaired autophagy triggers inflammatory response in macrophages. As a key transcription regulator, transcription factor EB (TFEB) activates the transcription of target genes related autophagy and lysosome by binding to promoters, whereas it is unclarified for the relationship between inflammatory response induced by cigarette smoke extract (CSE) and TFEB-mediated autophagy. Thus, we investigated the role of TFEB-mediated autophagy in inflammatory response induced by CSE in NR8383 cells, and to explore its potential mechanism. Methods: Based on cell viability and autophagy, cells treated with 20% concentration of CSE for 24 h were selected for further studies. Cells were divided into control group, chloroquine (CQ, the autophagy inhibitor) group, CSE group, CSE + rapamycin (the autophagy inducer) group and CSE + fisetin (the TFEB inducer) group. The levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in supernatant were detected by ELISA kits. The protein expressions were tested by western blot. The intensity of fluorescence of Lysosome-associated membrane protein 1 (LAMP1) and TFEB was detected by immunofluorescence. Lyso-Tracker Red staining was applied to detect the lysosome environment. Results: CSE inhibited the cell viability, increased the contents of TNF-α, IL-1β, IL-6, the ratio of LC3II/I, and the level of P62 protein. Besides, CSE decreased the fluorescence intensity of LAMP1 protein and Lyso-Tracker Red staining, as well as the ratio of nucleus/cytosol of TFEB protein. Activating autophagy with rapamycin alleviated CSE-induced inflammatory response. The activation of TFEB via fisetin alleviated CSE-induced autophagy impairment and lysosomal dysfunction, thus alleviated inflammatory response in NR8383 cells. Conclusion: CSE-induced inflammatory response in NR8383 cells, which may be related to the inhibition of TFEB-mediated autophagy.

Keywords:

• Alveolar macrophage
• autophagy
• cigarette smoke extract
• inflammation
• transcription factor EB

Introduction

Chronic obstructive pulmonary disease (COPD) is a group of preventable respiratory diseases, which is prominently characterized by chronic irreversible airflow restriction.¹ Chronic pulmonary inflammation caused by long-term smoking and inhalation of harmful particles is the core of complex pathogenesis of COPD.²

As the first cellular barrier against invasive pathogens, alveolar macrophages (AMs) are the important part in innate immune response.³ The abnormal function of macrophages is involved in the formation and aggravation of pulmonary inflammation of COPD, which might be closely related to the inflammation initiation, alveolar structure destruction and airway remodeling.⁴

Long-term smoking is one of the mainly risk factor of COPD. Modern studies have found that the dysfunction of AMs caused by cigarette smoke was involved in the formation and aggravation of pulmonary inflammation in COPD, which may be attributed to the increasing release of pro-inflammatory cytokines and chemokines.⁵ In addition, the phagocytosis of AMs exposed to cigarette smoke was weakened, thus the clearance rate of necrotic cells and bacteria was decreased, which led to the recruitment of inflammatory cells and bacterial colonization in the lung tissue.⁶

Autophagy is a cellular degradation pathway that maintains cell homeostasis by degradating damaged proteins and organelles via a lysosomal degradation pathway.⁷ As the important organelle for the degradation of biological macromolecules, lysosomes play a crucial role in autophagy degradation, which largely depends on its acidic environment.⁸ The dysfunction of lysosomes can inhibit the degradation stage of autophagy, leading to the accumulation of impaired proteins and organelles. In COPD patients, the ratio of autophagosome marker protein LC3II/I and the expression of autophagic substrate degradation protein P62 in the lung tissues and AMs significantly increased, which indicated that the degradation process of autophagy was impaired.^9–11 Besides, In vitro experiment, CSE impaired the degradation process of autophagy and increased the secretion of inflammatory cytokines, and activating impaired autophagy can inhibit the secretion of inflammatory cytokines.¹² These results suggested that CSE-induced autophagy impairment may be involved in the pulmonary inflammation of COPD.

As an important transcription regulator of autophagy-related and lysosomal-related genes, transcription factor EB (TFEB) activates the transcription of target genes by binding to specific DNA sites of gene promoters.¹³ Modern studies have found that the process of TFEB entering into nucleus was blocked in various disease models such as neurodegeneration and diabetic nephropathy, et al., and activating TFEB entering into nucleus significantly delayed the progression of diseases.^14–15

Recently, some researchers have found that the accumulation of perinuclear TFEB was positively related to the level of pulmonary inflammation in COPD, and activating TFEB significantly alleviated pulmonary inflammation, which indicated that TFEB may be a therapeutic target for COPD treatment.¹⁶ However, whether TFEB-mediated autophagy regulates inflammatory response induced by CSE in rats alveolar macrophages are still unclear. Therefore, we used NR8383 cells to verify the effect of CSE on autophagy and inflammatory response, and the mechanism of TFEB-mediated autophagy in CSE-induced inflammatory response.

Materials and methods

Cell culture and treatment

The rat alveolar macrophage cell line NR8383 cells (Shanghai Fuheng Biotech Co., Ltd, No. FH0398) were cultured in F-12K medium with15% fetal bovine serum and 1% PS. Cigarette smoke extract (CSE) was made by burning three The Lion cigarettes (11 mg coke, 1.0 mg nicotine, 11 mg carbon monoxide; Anhui Tobacco Industry Co., China) and the smoke was dissolved in serum-free F-12K medium (20 mL). An OD (320 nm) of 0.74 was considered to be 100% CSE, and F-12K medium was used to adjust CSE working concentration.¹⁷ To investigate the effect of CSE on cell viability, cells were treated with different concentrations of CSE (5%, 10%, 20%, 40%) for 24 h, 48 h, 72 h, respectively, then the viability was measured by CCK-8 solution.

To explore the role of autophagy impairment in inflammatory response in NR8383 cells, cells in CQ group were treated with autophagy inhibitor chloroquine (CQ) (MedChem Express [MCE], No.HY-17589A, 40 μM) for 24 h. To investigate the function of CSE-induced autophagy impairment in inflammatory response, cells in CSE + RAPA group were pretreated with autophagy inducer-rapamycin (RAPA) (MCE, No.HY-10219, 3 μM) for 30 mins.¹² Similarly, to examine the potential role of TFEB in CSE-induced inflammatory response, cells in CSE + fisetin group were pretreated with fisetin (MCE, No.HY-N0182, 20 μM) for 30 mins.¹⁸

Cell viability assay

Cell Counting Kit-8 (CCK8) (Bioss, No. BA00208) was used to assess cell viability. In brief, NR8383 cells were inoculated into 96-well plates at a density of 2 × 10⁹/L. Then cells were stimulated with 5%, 10%, 20% and 40% CSE for 24 h, 48 h and 72 h, respectively. After treatment, CCK8 solution was added to each well, with 10 µL a well. After incubation for 2 h in the dark, the absorbance value of each well was tested with a microplate reader at 450 nm (Tecan, Shanghai, China).

Enzyme-linked immunosorbent assay (ELISA)

The cell suspension was centrifuged at 600 g at 4 °C for 5 min, and the supernatant was used to detect the contents of TNF-α, IL-1β, and IL-6 by ELISA kits (No. JYM0646Ra, JYM0635Ra, JYM0419Ra; Wuhan Jiyinmei Biotech Co., Wuhan, China).

Western blot

The nuclear and cytoplasmic protein extraction kit (P0028, Beyotime, Shanghai, China) was used to extract nuclear and cytoplasmic proteins. RIPA lysis buffer and protease inhibitors (No. P0013B, Beyotime, Shanghai, China) were used to collect total protein. 30 μg samples were added to the wells of SDS-PAGE (8%–12%) to separate proteins, and then separated samples were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA). The nonspecific binding sites of membranes were blocked with 5% BSA at room temperature for 1 h, then added primary antibodies against LC3B (No.bs-4843R, Bioss, 1:500), P62 (No.bs-2951R, Bioss,1:1000), GADPH (No. TA-08, Zs-BIO, 1:1000), Lamin B (No. bs-24328R, Bioss, 1:1000), and TFEB (No. bs-5137R, Bioss, 1:1000) to the incubator box to incubate with the membranes at 4 °C overnight, respectively. Then, secondary antibody (No. Bs-0295G, Bioss, 1:15000) was added to incubate with the membranes at room temperature. Immunoreactive protein bands were visualized by chemiluminescence kit (No. 32134, Thermo). Image J software was used to quantify immunoblots and normalized by housekeeping protein GADPH (for nuclear protein, housekeeping protein is Lamin B).

Immunofluorescence

NR8383 cells were collected and fixed with 4% formaldehyde for 20 min at 4 °C, permeabilized with 0.2% Triton X-100 for 15 min, then blocked with 5% BSA at room temperature for 1 h. Then cells were incubated with primary antibody against LAMP1 (No.bs-1970R, Bioss, 1:200), TFEB (No.bs-5137R, Bioss, 1:300) at 4 °C overnight, respectively. The primary antibody was removed and the secondary antibody (No. bs-0295G-FITC, Bioss, 1:500) was incubated for 1 h in the dark. Then anti-fluorescence quench agent containing DAPI was added to the samples. The fluorescence microscope (Leica, Germany) was used to collect images.

Lyso-Tracker red staining

NR8383 cells were plated into 24-well plates. After intervation, the original medium was discarded and cells were washed with 1 × PBS for three times, then cells were incubated in the medium containing 50 nmol/L Lyso-Tracker red (Beyotime, No. C1046) for 30 min in the cell incubator. Washing with PBS for three times, lysosomal staining was observed using fluorescence microscope.

Transmission electron microscopy

Autophagosomes in cells were observed by TEM. Briefly, the samples were fixed with 2.5% glutaraldehyde at 4 °C overnight, then fixed with 2% osmic acid for 1 h, and dehydrated in a gradient concentration of alcohol before being embedded. Ultrathin section with thickness of 70 nm were cut using a Leica ultracut ultramicrotome (Leica EM UC7, Germany), then the autophagosome in the samples were observed by electron microscopy (Tecnai G2 20 S-Twin, USA).

Statistics

SPSS23.0 software was used to perform statistical analysis. Data were presented as mean ± SEM. One-way ANOVA was applied to analyze the significance of statistic between multiple groups, and least-significant difference (LSD) test was applied to pairwise comparisons with homogeneity of variance.

Results

CSE inhibited the cell vitality of NR8383 cells

As the results showed, CSE inhibited the vitality of NR8383 cells in a concentration- dependent manner (P < 0.05 P < 0.01, Figure 1). Compared with the 24 h group, the cell viability of 48 h group, 72 h group significantly decreased (P < 0.05, P < 0.01, Figure 1). Besides, there was no significant difference between 48 h group and 72 h group (P > 0.05, Figure 1). In addition, the cell viability of 24 h group was at a fine level (Figure 1). Therefore, 24 h was selected as the CSE intervention time in the following experiments.

Figure 1. CSE inhibited the viability of NR8383 cells. The data was expressed as mean ± SEM (n=3); *P<0.05, **P<0.01, ***P<0.001 vs control group.

CSE-induced autophagy impairment and inflammatory response in NR8383 cells

LC3 is a recognized marker of autophagy. When autophagy is activated, LC3-I is gradually transformed into LC3-II, which locates on the membranes of autophagosomes. Therefore, the ratio of LC3II/I protein is positively correlated with the number of autophagosomes. As an autophagy substrate protein, P62 is widely used as an index of autophagy degradation, which is negatively correlated with autophagy. To investigate whether CSE-induced autophagy impairment in NR8383 cells, we detected the ratio of LC3II/I protein and the level of P62 protein, as well as the number of autophagosomes.

As the results showed, CSE increased the ratio of LC3II/I protein and the levels of P62 protein in a concentration-dependent manner (P < 0.05, P < 0.01, P < 0.001, Figure 2A). Besides, in the control group, there were rare autophagosomes in NR8383 cells; whereas, in the CSE group, autophagosomes were clearly visible and abundant (Figure 2B). These results revealed that CSE impaired the degradation process of autophagy in a concentration-dependent manner. Considering the cell viability of 20% CSE stimulation for 24 h was 70.17 ± 1.68 (%), at a great cell viability (Figure 1), we selected 20% CSE, 24 h as the concentration and time of CSE intervention to further investigate the role of TFEB-mediated autophagy in inflammatory response in NR8383 cells.

Figure 2. CSE induced autophagy impairment and inflammatory response in NR8383 cells. A: The ratio of LC3II/I protein, the level of P62 protein in NR8383 cells were detected by western blot. B: autophagosomes in NR8383 cells were detected by TEM (the yellow arrows indicated autophagosomes). C: Immunofluorence (IF) was applied to detect the fluorescence intensity of LAMP1 protein in NR8383 cells. Scale bar, 20µm. D: Lyso-Tracker Red staining was used to measure the acid environment of lysosomes in NR8383 cells; the intensity of red fluorescence was positively correlated with the acidic degree of lysosomes. Scale bar, 20µm. E: The levels of TNF-α, IL-1β, IL-6 were detected by ELISA kits. The data was expressed as mean ± SEM (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. control group.

Lysosomes are organelles for the degradation stage of autophagy, and the degradation function of it largely depends on its acidic environment. Lysosome-associated membrane protein 1 (LAMP1), a highly glycosylated protein, mainly locates on lysosomal membranes, which not only maintains the structural integrity of lysosomes but acid environment in lysosomes.¹⁹ As the result showed, compared with the control group, the fluorescence intensity of LAMP1 protein and Lyso-Tracker Red staining in the CSE group significantly decreased (P < 0.01, Figure 2C&D), revealing that the relative level of lysosomes was decreased and the acidic environment of lysosomes was destroyed, respectively. These results indicated that lysosomes were impaired in the CSE group, which may be involved in the impairment of autophagy degradation.

To observe the effect of CSE on the inflammatory response in NR8383 cells, we detected the contents of inflammatory cytokines. As the results showed, comparing to the control group, the contents of inflammatory cytokines TNF-α, IL-1β and IL-6 in the CSE group were notably increased (P < 0.001, Figure 2E), which indicated that CSE-induced inflammatory response in NR8383 cells.

CSE-induced inflammatory response via preventing TFEB from entering into nucleus in NR8383 cells

To further investigated whether CSE-induced autophagy impairment triggered inflammatory response in NR8383 cells, autophagy inhibitor CQ and autophagy activator RAPA were used. Comparing to the control group, the ratio of LC3II/I protein, the level of P62 protein and the levels of TNF-α, IL-1β, and IL-6 in the CQ group notably increased (P < 0.01 and P < 0.001, Figure 3A&B). These results suggested that autophagy impairment can induce inflammatory response in NR8383 cells. Comparing with the CSE group, the ratio of LC3II/I protein notably increased, while the level of P62 protein and TNF-α, IL-1β, and IL-6 significantly decreased in CSE + RAPA group (P < 0.05, P < 0.001, Figure 3A&B). These results revealed that CSE-induced autophagy impairment was involved in the inflammatory response in NR8383 cells, and activating impaired autophagy could partly reverse CSE-induced inflammatory response.

Figure 3. CSE prevented TFEB from entering into nucleus, which led to the increasing secretion of inflammatory cytokines in NR8383 cells. A: The ratio of LC3II/I protein, the level of P62 protein in NR8383 cells were detected by western blot. B: The levels of inflammatory cytokines TNF-α, IL-1β, IL-6 were detected by ELISA kits. C: The immunofluorence was used to analyze the TFEB location in NR8383 cells. Scale bar, 20µm. D: The expressions of TFEB protein in nucleus and cytosol were detected by western blot. E: Immunofluorence (IF) was used to detect the fluorescence intensity of LAMP1 protein in NR8383 cells. Scale bar, 20µm. F: Lyso-Tracker Red staining was used to measure the acid environment of lysosomes in NR8383 cells; the intensity of red fluorescence was positively correlated with the acidic degree of lysosomes. Scale bar, 20µm. The data was expressed as mean ± SEM (n=3). *P<0.05, **P<0.01, ***P<0.001 vs control group, #P<0.05, ##P<0.01, ###P<0.001 vs CSE group.

TFEB is an important transcriptional regulator of autophagy and lysosomal biosynthesis. When activated, TFEB enters into the nucleus and binds to its target gene promoters to promote the transcription of target genes. Firstly, we investigated the location of TFEB protein in NR8383 cells. As the results showed, comparing to the control group, the nuclear location of TFEB protein and the ratio of nucleus/cytosol of TFEB protein in the CSE group notably decreased (P < 0.01 and P < 0.001, Figure 3C&D).

To further verify whether TFEB was involved in CSE-induced autophagy impairment, we pretreated NR8383 cells with fisetin, a TFEB inducer, for 30 min, and then treated with 20% CSE for 24 h. As the results showed, compared with the CSE group, the nuclear location of TFEB protein and the ratio of nucleus/cytosol of TFEB protein in the CSE + fisetin group notably increased (P < 0.05, Figure 3C&D), and the ratio of LC3II/I protein and the level of P62 protein, as well as the levels of TNF-α, IL-1β, and IL-6 decreased (P < 0.05, P < 0.001, Figure 3A&B). Whereas, comparing to the CSE group, the fluorescence intensity of LAMP1 protein and Lyso-Tracker Red staining in the CSE + fisetin group notably increased (P < 0.05, Figure 3E&F). These results indicated that CSE can inhibit the degradation process of autophagy by preventing TFEB from entering into the nucleus, thus further induce inflammatory response in NR8383 cells.

Discussion

The prevalence of COPD is increasing year by year, with more than 5.4 million people likely to die from COPD and related diseases annually by 2060.²⁰ The pathogenesis of COPD is complex, up to now, persistent lung inflammation is still considered to be the core mechanism of COPD.²¹ Long-term smoking has been considered as the major risk factor of COPD, which is closely related to the persistent lung inflammation in COPD patients.²² Many studies have verified that CS can activate inflammatory cells by triggering the release of inflammatory cytokines and pattern recognition receptors such as Toll-like receptor 4 (TLR4), thus leading to chronic lung inflammation.²³ CSE is commonly used to mimic the conditions of inflammatory response in smokers in vitro studies, thus we stimulated NR8383 cells with CSE to prepare vitro model. Our results indicated that CSE increased the secretion of inflammatory cytokines TNF-α, IL-1β, and IL-6 in NR8383 cells.

Autophagy, a “self-eating” catabolic process, is markedly essential to maintain cell homeostasis and normal cellular function.²⁴ Previous study has demonstrated that autophagy was impaired in the lung of mice exposed to chronic/sub-chronic cigarette smoke, which was associated with the aggravation of alveolar senescence, COPD- emphysema.²⁵ Besides, some researchers also found that CSE can induce impaired autophagy in the human bronchial epithelial cells, which can activate cellular apoptosis and senescence.^26–27 These results suggested that the impaired autophagy was closely related to the pathogenesis of COPD.

As the first cellular barrier against invasive pathogens, AMs are the important part in innate immune response. The abnormal functions of AMs were involved in the formation and aggravation of pulmonary inflammation of COPD. Recent studies have demonstrated that autophagy was involved in the regulation of macrophage.^28–29 Therefore, we verified the effect of CSE on autophagy in NR8383 cells and its specific mechanisms.

LC3 is a recognized autophagy specific marker. With the modification of autophagy-related protein 4 (ATG4), LC3-I gradually transforms into LC3-II, which is located on the membranes of autophagosomes. Therefore, the ratio of LC3II/I protein can reflect the number of autophagosomes.³⁰ In our study, we found that in the CSE group, the number of autophagosomes and the ratio of LC3II/I protein markedly increased in NR8383 cells, whereas autophagy is a dynamic process including the following steps: initiation of autophagy, extension of autophagosome membrane, formation of autophagy-lysosome and degradation of lysosome.³¹ Therefore, the increasing number of autophagosomes may be related to the activation of autophagy, or the abnormal degradation of autolysosomes. Thus, we further investigated the expression of autophagy substrate protein P62, which is negatively correlated with autophagy. Our results indicated that the ratio of LC3II/I protein and the level of P62 protein significantly were increased in the CSE group. Combined with the increasing number of autophagosomes in the CSE group observed by TEM, we believed that CSE mainly affected the degradation stage of autophagy, resulting in the accumulation of impaired proteins and organelles.

Lysosomes are organelles for degradation of autophagy substrates, the degradation function of lysosomes largely depends on its acidic environment.³² Therefore, we further investigated the function of lysosomes of NR8383 cells in the CSE group. LAMP1 is the marker protein of lysosomes, which not only maintains the structural integrity of lysosomes but acid environment.³³ As the results showed, compared with the control group, the fluorescence intensity of LAMP1 protein and Lyso-Tracker Red staining significantly decreased in the CSE group. These results indicated that CSE impaired the function of lysosomes in NR8383 cells.

It has been proved that autophagy plays an important role in the regulation of macrophage functions, the accumulation of impaired proteins and organelles caused by autophagy disorder can lead to the dysfunction of macrophage phagocytosis and antigen presentation, resulting in the increased secretion of inflammatory cytokines.³⁴ To further investigate whether CSE-induced autophagy impairment triggered inflammatory response in NR8383 cells, rapamycin(RAPA), an autophagy activator, was used to pretreat cells before CSE stimulation. The results showed that RAPA partially reversed CSE-induced autophagy impairment and decreased the contents of TNF-α, IL-1β, and IL-6.

TFEB is a key molecule that regulates autophagy and lysosomal biosynthesis at the transcriptional level, its helix-loop-helix-leucine zipper domain (HLH-leucine zipper domain) can bind to specific DNA to initiate the transcription of target genes.³⁵ Under physiological conditions, TFEB is located in cytoplasms as non-activated phosphorylation, and under stress conditions, such as starvation, lysosome damage, etc., TFEB is activated and dephosphorized, rapidly enters into nucleus, then binds to specific sites of target gene promoters to initiate transcription of target genes.³⁶ In recent years, with the further study of TFEB, autophagy and lysosomal dysfunction induced by preventing TFEB from entering into nucleus has been involved in the development of many diseases.^37–39 Similarly, some studies have identified that CSE can inhibit the nuclear translocation of TFEB^16,18 In this study, our results indicated that the nuclear localization of TFEB was decreased in the CSE group, and TFEB inducer fisetin activated TFEB entering into the nucleus, thus alleviated CSE-induced autophagy impairment and lysosomal dysfunction.

As an antioxidant, fisetin is widely used to improve brain health. In recent studies, fisetin has been shown to activate TFEB and consequentially activate impaired autophagy.^16,40 Similarly, as our results showed, activating TFEB entering into nuclei with fisetin can activate impaired autophagy induced by CSE in NR8383 cells, thus alleviate inflammatory response. These results suggest that fisetin provides a new therapeutic method for the treatment of lung inflammation by targeting the mechanism of autophagy in inflammatory response.

Previous studies have verified that CSE can impair the degradation process of autophagy in COPD animal models, human tissues and macrophages^9,12. In our study, we revealed that CSE can impair the degradation process of autophagy in NR8383 cells, thus leading to the inflammatory response, which was consistent with previous studies. Besides, our study indicated that CSE can prevent TFEB from entering into nuclei, which resulted in the impairment of autophagy and lysosome function in NR8383 cells, whereas in this study, we only verified the conclusion in rat alveolar macrophage NR8383 cells and did not verify the conclusion in other alveolar macrophage cell lines. Besides, to further explore the pathogenesis of COPD, we should further investigate whether TFEB-mediated autophagy is impaired in alveolar macrophages in COPD patients and animal models and the relationship between TFEB-mediated autophagy and pulmonary inflammation, so as to provide a new target for clinical treatment of COPD.

Disclosure statement

No conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 81373743), Natural Science Foundation of Anhui Province in China (No. 2008085MH267), Key Project of Natural Science of Anhui Provincial Department of Education [2022AH040078], and Natural Science Foundation of the Anhui University of Chinese Medicine (No. 2020zrzd03).