Transcription Factor EB Alleviates Endothelial Cell Inammation Through NLRP3 Inammasome-Mediated Cell Pyroptosis in Atherosclerosis

Increasing evidence suggests that transcription factor EB (TFEB) inhibits inammation in endothelial cell (ECs) and reduces development of atherosclerosis. However, little is known about the mechanism of action of TFEB on inammation in atherosclerosis (AS). The levels of TFEB, NLRP3, VCAM-1, ICAM-1, E-selectin, MCP-1, cleaved caspase-1, IL-1β and IL-18 in ECs were examined by immunoblotting, quantitative real time-polymerase chain reaction (qRT-PCR) , Enzyme-linked immunosorbent assay. The LDH activity were examined by LDH assay. TUNEL-positive cell were examined by TUNEL assay. The relationship between TFEB and NLRP3 were examined by immunouorescence and coimmunoprecipitation. The effects of TFEB on atherosclerotic lesions by hematoxylin and eosin, TUNEL and collagen staining in the aortic valve of ApoE -/- mice fed a high fat diet (HFD). Here, we report that H 2 O 2- induced cell pyroptosis and inammatory response were mainly due to nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inammasome activation. The nuclear protein TFEB was signicantly increased by H 2 O 2 , and knockdown of TFEB aggravated cell pyroptosis and inammatory response. TFEB directly bound to NLRP3 and blocked NLRP3-mediated cell pyroptosis and inammatory response. The effect of H 2 O 2 on TFEB might be associated with AMP-activated protein kinase/mechanistic target of rapamycin-dependent signaling pathways.


Introduction
It has been shown that oxidative modi cation of lipids and proteins is detectable in the vascular lesions of atherosclerosis (AS) and the degree of oxidation correlates closely with the severity of the AS 1 .
Oxidative-stress-induced injury of endothelial cells (ECs) is one of the main factors in AS, which stimulates the secretion of in ammatory factors and triggers the accumulation of in ammatory factors in ECs, leading to the formation of atherosclerotic plaques [2][3][4][5] . Many studies have revealed that antioxidants possess therapeutic bene ts to combat the progression of cardiovascular disease 6 .
Therefore, reducing oxidative-stress-induced in ammation may be bene cial in preventing the progression of atherosclerosis.
Pyroptosis, also known as in ammatory cell death, is a form of programmed cellular necrosis in which cells continue to swell until the cell membrane ruptures, resulting in the release of cellular contents and activation of a severe in ammatory response. Studies have indicated that pyroptosis plays a key role in the pathogenesis of cardiovascular disease. More importantly, it was found that NLRP3-related downstream signaling molecules (apoptosis-associated speck-like protein containing a CARD [ASC], caspase-1, interleukin [IL]-1β and IL-18) were signi cantly more expressed in atherosclerotic plaque than in non-atherosclerotic vessels, and higher expression of caspase-1, IL-1β and IL-18 was closely correlated with atherosclerotic plaque fragility, suggesting that activation of the NLRP3 in ammasome mediated the evolution of atherosclerotic lesions 7 . The development of cell pyroptosis is closely related to activation of the NLRP3 in ammasome, which is one of the major in ammatory complexes in cell death.
Therefore, research on the therapeutic targets of NLRP3 in ammatory corpuscles and pyroptosis is important for the prevention and treatment of cardiovascular diseases 8 . However, the exact mechanism of action of pyroptosis on AS is still unclear.
Transcription factor EB (TFEB) is a member of the microphthalmia/transcription factor E (MiT/TFE) family of basic helix-loop-helix leucine zipper transcription factors. TFEB is involved in the development of neurodegenerative diseases, cancer and atherosclerotic diseases. TFEB is the master regulator governing gene regulation of autophagy and lysosomal biogenesis, and is involved in many pathophysiological processes, such as mitochondrial function, lipid and energy metabolism, and in ammation 9,10 . Additionally, knockdown of TFEB leads to accumulation of lipid droplets and impairment of lipid degradation 11 . Recently, increased evidence has indicated that TFEB acts as protective factor in AS through inhibition of EC in ammation 9,12 . Whether TFEB is involved in AS, and its mechanism of action on cell pyroptosis remain to be clari ed.
Here, our novel nding con rmed that TFEB inhibited cell pyroptosis induced by NLRP3, which attenuated the H 2 O 2 -induced in ammatory response in ECs. The mechanism of H 2 O 2 regulation of TFEB was mainly due to the mechanistic target of rapamycin (mTOR)-dependent signaling pathways. We explored the role of TFEB and its mechanisms in mediating the NLRP3 in ammasome and cell pyroptosis in the pathogenesis of AS.

Results
Oxidative stress triggers pyroptosis and in ammatory response in ECs. H 2 O 2 can induce an in ammatory response though triggering oxidative stress in cardiovascular disease 13,14 . Recent studies have shown that pyroptosis plays key roles in the in ammatory response. Therefore, we assessed the relationship between H 2 O 2 and cell pyroptosis and in ammatory response in ECs. In ECs, H 2 O 2 exposure (0, 25, 50 or 100 μmol) increased the in ammatory response as indicated by a dose dependent increase of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, E-selectin and monocyte chemoattractant protein (MCP)-1 expression and production ( Figure  1A-C). These results showed that H 2 O 2 induced an in ammatory response in ECs. We investigated the effect of H 2 O 2 on cell pyroptosis. In ECs exposed to H 2 O 2 , the level of cleaved caspase-1, IL-1β and IL-18 was signi cantly increased in a dose-dependent manner ( Figure 1D and E), which was accompanied by an increase in lactate dehydrogenase (LDH) activity ( Figure 1F). This suggested that H 2 O 2 promoted cell pyroptosis. Collectively, our results revealed that H 2 O 2 -induced cell pyroptosis may contribute to the in ammatory response in ECs.
NLRP3 in ammasome contributes to cell pyroptosis under oxidative stress in ECs.
Many studies have shown that the biochemical function of the NLRP3 in ammasome is to activate caspase-1, which leads to maturation of IL-1β and IL-18 and induction of cell pyroptosis 15  suggesting that NLRP3 aggravated cell pyroptosis and in ammatory response induced by H 2 O 2 ( Figure   2P). These results indicated that NLRP3 contributed to cell pyroptosis by releasing proin ammatory cytokines under oxidative stress.
TFEB is required for pyroptosis and in ammatory response induced by NLRP3 under oxidative stress in ECs.
TFEB in ECs inhibits in ammation and reduces AS development 16 . To further elucidate the essential role of TFEB in regulating pyroptosis and in ammatory response in ECs, we used siRNA to inhibit expression of TFEB ( Figure S1C). TFEB knockdown signi cantly enhanced expression and production of VCAM-1, ICAM-1, E-selectin and MCP-1 ( Figure 3A-C), suggesting that TFEB inhibited the in ammatory response. TFEB-shRNA signi cantly enhanced NLRP3, cleaved caspase-1, IL-1β and IL-18 ( Figure 3D and E) expression, along with LDH activity ( Figure 3F) and TUNEL-positive cells ( Figure 3G). Overexpression of TFEB ( Figure S1D) signi cantly attenuated NLRP3, cleaved caspase-1, IL-1β and IL-18 expression ( Figure  3H and I) and LDH activity ( Figure 3J). These results indicated that TFEB alleviated cell pyroptosis and in ammatory response. We investigated whether NLRP3 involvement in pyroptosis and the in ammatory response was inhibited by TFEB. Knockdown of NLRP3 attenuated TFEB-shRNA-induced expression and production of VCAM-1, ICAM-1, E-selectin and MCP-1 ( Figure 3K-M). Subsequently, knockdown of NLRP3 attenuated TFEB-shRNA-induced expression of cleaved caspase-1, IL-1β and IL-18 in ECs ( Figure 3N and O). NLRP3-shRNA treatment decreased TFEB-shRNA-induced LDH activity ( Figure 3P), and TFEB-shRNAinduced TUNEL-positive cells were further increased by pcDNA-NLRP3 ( Figure 3Q). The results con rmed that TFEB alleviation or pyroptosis and the in ammatory response were mainly due to NLRP3 inhibition in ECs. We elucidated the possible role of TFEB in H 2 O 2 -induced pyroptosis and in ammatory response in ECs. Cytoplasmic TFEB was markedly decreased and nuclear TFEB was increased by H 2 O 2 in a dosedependent manner ( Figure 3R). Overexpression of TFEB markedly suppressed H 2 O 2 -induced VCAM-1, ICAM-1, E-selectin and MCP-1 level and production ( Figure 3S-U), suggesting that TFEB alleviated the in ammatory response induced by H 2 O 2 in ECs. Overexpression of TFEB inhibited H 2 O 2 -induced expression of NLRP3, cleaved caspase-1, IL-1β and IL-18 ( Figure 3V and W). In accordance with reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting results, LDH activity induced by H 2 O 2 was decreased by pcDNA-TFEB ( Figure 3X). The number of TUNEL-positive cells induced by H 2 O 2 was also further increased by TFEB-shRNA ( Figure 3Y). These results indicated that TFEB suppressed NLRP3 in ammasome expression, which alleviated cell pyroptosis and in ammatory response induced by oxidative stress, suggesting that TFEB exerted a potent protective effect on ECs in the presence of oxidative stress.

TFEB interacts with NLRP3
The above results demonstrated that nuclear translocation of TFEB increased under oxidative stress and TFEB alleviated the NLRP3 levels in ECs. To establish the mechanism of action of TFEB on NLRP3 in AS, we measured colocalization of TFEB and NLRP3 in the presence of H 2 O 2 , using immuno uorescence confocal microscopy. Colocalization of TFEB and NLRP3 was detected in ECs when exposed to H 2 O 2 ( Figure 4A). We carried out coimmunoprecipitation using an anti-NLRP3 antibody, followed by western blotting of the expression of TFEB and NLRP3. We demonstrated the presence of TFEB and NLRP3 in the immunoprecipitation group compared with the IgG group, con rming an interaction between TFEB and NLRP3 ( Figure 4B). The analysis showed that TFEB interacts with NLRP3.
AMP-activated protein kinase (AMPK)/mTOR signaling pathway contributes to TFEB-mediated cell pyroptosis and in ammatory response under oxidative stress.
The above results con rmed that TFEB inhibited NLRP3 in ammasome activation and cell pyroptosis. A recent study showed that the AMPK/mTOR pathway serves as a novel regulator of cell fate during differentiation via TFEB-dependent regulation of autophagic ux 17,18 . We investigated whether this was also the case in H 2 O 2 -induced pyroptosis. Phosphorylation of AMPK was increased, whereas phosphorylation of mTOR was decreased by H 2 O 2 in a dose-dependent manner, with no change in AMPK and mTOR total protein expression levels ( Figure 5A). However, AMPK phosphorylation was decreased and mTOR phosphorylation was increased after AMPK inhibition (Compound C; CC) (Figure.  Figure 5E and F) along with LDH activity ( Figure 5G) and number of TUNEL-positive cells ( Figure 5H). We also found that H 2 O 2 -induced TFEB nuclear protein was inhibited by CC ( Figure 5I). Conversely, cytoplasmic TFEB inhibition by H 2 O 2 was reversed by CC, suggesting that the level of TFEB was regulated by H 2 O 2 partly through the AMPK/mTOR signaling pathway. These results suggested that H 2 O 2 regulated the TFEB expression partly through the mTOR-dependent signaling pathway in ECs.
To investigate whether TFEB plays a role in the pathogenesis of AS, we explored the effects of TFEB on atherosclerotic lesions by hematoxylin and eosin staining in the aortic valve of ApoE -/mice fed a high fat diet (HFD). Expression of TFEB in the HFD group was decreased, whereas NLRP3 expression was increased ( Figure 6A). TFEB-shRNA induced cell death in ApoE -/mice treated with HFD ( Figure 6B). As expected, the lesion areas and collagen content in the aortic valve were increased in the HFD group, and TFEB-shRNA aggravated the changes in lesion areas and collagen content caused by HFD ( Figure 6C).
The above ndings indicated a protective role of TFEB against the development of vulnerable atherosclerotic plaques.  23 . In agreement with previous study, our results showed that the in ammatory response was increased by H 2 O 2 , and H 2 O 2 induced the activation of pyroptosis in ECs. Our results also showed that the in ammatory response induced by H 2 O 2 was mainly due to the activation of cell pyroptosis, indicating that H 2 O 2 -induced pyroptosis might be a cellular mechanism for the in ammatory response in AS. Therefore, the underlying molecular mechanism of oxidative stress in in ammation, except pyroptosis, requires further exploration.

Discussion
The current study demonstrated that the NLRP3 in ammasome was closely related to cell pyroptosis and plays a crucial role in cardiovascular disease. Numerous studies have shown that components of the NLRP3 in ammasome signaling pathway (NLRP3, ASC, caspase-1, IL-1β and IL-18) are predominantly localized in human carotid unstable atherosclerotic plaques 24 . In the mouse myocarditis model, 1,25dihydroxyvitamin D3 can inhibit the cardiomyocyte pyroptosis signaling pathway and prevent cardiomyocyte death, and then improve the lesions of myocarditis 25 . The miRNA-9 acts on the protein ELAV-like protein 1, which in turn inhibits expression of caspase-1 and IL-1β in cardiomyocytes, and inhibits high glucose-induced cardiomyocytes pyroptosis 26 . Activation of NLRP3-in ammasome-regulated pyroptosis can aggravate myocardial ischemic injury 27 . We found that oxidative-stressmediated NLRP3 in ammasomes play an important role in the development of AS. Moreover, expression of NLRP3 was upregulated by H 2 O 2 , and H 2 O 2 aggravated cell pyroptosis and the in ammatory response.

NLRP3 enhanced cell pyroptosis and the in ammatory response induced by H 2 O 2 . A previous study has
revealed that lipopolysaccharide can aggravate hypoxia/reoxygenation and high glucose-induced cardiomyocyte injury by activating reactive oxygen species (ROS)-dependent NLRP3-mediated pyroptosis 28 . Nicotine promotes the development of AS through ROS/NLRP3-regulated EC pyroptosis 29 .
These results indicated that oxidative stress triggered NLRP3-induced cell pyroptosis, resulting in an in ammatory response and cell damage, but the exact mechanism of action of oxidative stress in cell pyroptosis needs further investigation.
TFEB is a master regulator of lysosome biogenesis and autophagy. TFEB overexpression acts against AS by reducing production of the proin ammatory cytokine IL-1β and enhancing cholesterol e ux in macrophages. Numerous studies have shown that the disaccharide trehalose acts as a novel inducer of TFEB with similar atheroprotective effects 30 . TFEB has a transcriptional regulatory function during lipid catabolism though proliferator-activated receptor γ coactivator-1α. TFEB represents a novel resveratrol target related to the protection against EC oxidative damage 31 . In agreement with a previous study, we found that TFEB alleviated the in ammatory response in ECs. TFEB also attenuated NLRP3-dependent cell pyroptosis and in ammatory response. These effects of TFEB were further reversed by NLRP3. Nuclear TFEB was upregulated by H 2 O 2 and alleviated cell pyroptosis and in ammatory response.
Previous studies have shown that overexpression of TFEB suppresses endothelial in ammation and attenuates AS in mice 32 . Collectively, these results indicate that TFEB protects cells from oxidative stress damage. Consistently, in our study, the lesion area was further enhanced in ApoE / mice fed an HFD by knockdown of TFEB. The above results provided evidence for the underlying mechanisms of the antiin ammatory effects of TFEB, in which inhibition of NLRP3-induced pyroptosis may play a key role. However, trehalose activates autophagy and inhibits the activity of the in ammasome via TFEB, and ultimately alleviates the development of AS 30 . Jiang et al. showed that acrolein inhibits EC migration by inducing NLRP3-regulated pyroptosis through ROS-dependent autophagy 33 . The detailed mechanisms of TFEB in pyroptosis by autophagy will be further explored in future studies.
Recent studies have provided evidence that TFEB is regulated by mTOR in the presence of nutrients 34 . The mTOR-dependent pathway is a pivotal mechanism to strengthen the function of TFEB. The phosphorylates and nuclear fraction of TFEB are increased by extracellular signal-regulated kinase 2 35,36 . The deacetylation effect of SIRT1 and the AMPK/SIRT1 pathway play a critical role in TFEBregulated gene transcription 37 . We found that H 2 O 2 -regulated TFEB expression was mainly due to the mTOR-dependent signaling pathway. Moreover, the TFEB/NLRP3 pathway was associated with mTORdependent signaling pathways regulated by H 2 O 2 , which veri ed the involvement of these pathways in pyroptosis. The detailed mechanisms of action of H 2 O 2 in TFEB will be further explored in future studies.
As shown in the schematic diagram in Figure 7, our results provided evidence that TFEB suppressed NLRP3 in ammasome expression and cell pyroptosis, which alleviated the in ammatory response induced by oxidative stress. Moreover, the TFEB suppressed by H 2 O 2 was mainly associated with mTORdependent signaling. These novel nding are relevant to enhancing our understanding of the pathogenesis of AS and reveal that TFEB could act as novel potential therapeutic target.

Cell culture
Human umbilical vein ECs (ATCC CRL-1730) were purchased from American Type Culture Collection (Manassas, VA, USA. The ECs were seeded in DMEM provided with 10% fetal bovine serum at 37°C with 95% air and 5% CO 2 . Cells were grown in 6-or 12-well plates or 60-mm dishes and grown to 70%-80% con uence before use.

Western blotting and immunoprecipitation analysis
Protein lysates were separated by 12.5% SDS-PAGE. The western blots were incubated for 12 h with 1:800-diluted primary antibodies, and then incubated for 2 h at room temperature with secondary antibodies. The chemiluminescence of proteins was used by ECL Plus Western Blot Detection Sy stem (Amersham Biosciences, Foster City, CA, USA). Immunoprecipitation analysis was performed as previously described 38 . EC lysates were precleared by a conjunction of 1 mg control IgG and protein A/G PLUS-agarose beads, and then incubated overnight at 4°C with anti-NLRP3 antibodies, with addition of 25 ml protein A/G PLUS-agarose beads. The resulting immunoprecipitates were dissolved in SDS-PAGE sample buffer for electrophoresis and immunoblot analysis.
Quantitative RT-PCR Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed in cultured cells. Real-time PCR was carried out on the LightCycler 480 II (Roche, Pleasanton, CA, USA) with SYBR Green Dye detection (TaKaRa Bio, Mountain View, CA, USA). GAPDH was used for normalization.
All samples were assayed in triplicate and the data were analyzed using the ΔΔCt method. The primer sequences are listed in Supplementary Table 1.

Recombinant plasmid construction
The full-length TFEB and NLRP3 cDNA plasmid was obtained from OriGene (Rockville, MD, USA). TFEB cDNA was magni ed by PCR and subcloned into the pcDNA3.1(+) vector. The correct sequence of the TFEB and NLRP3 cDNA was con rmed by sequencing and named pcDNA-TFEB/NLRP3 in the recombinant plasmid.
The siRNA assay Control siRNA (negative control), siRNA-TFEB and siRNA-NLRP3 were purchased from Ribo Targets (Guangzhou, China). Control samples for all experimental procedures were processed with a nontargeting control mimic sequence of equal concentration. The e ciency of siRNA protein were veri ed by western blotting. The sequences of TFEB-shRNA, NLRP3-shRNA and control siRNA are listed in Supplementary Table 2.

LDH
The release of LDH can be used to assess cell pyroptosis 39 . The LDH-cytotoxicity Assay Kit was used to detect LDH release (Nanjing Jiancheng Biology Engineering Institute, Nanjing, Jiangsu, China).

Enzyme-linked immunosorbent assay
The concentrations of VCAM-1, ICAM-1, E-selectin and MCP-1 in EC culture supernatants in human serum samples were measured by ELISA kits. These kits were purchased from Abcam. TUNEL TUNEL assays for cell and tissue sections were performed using an In Situ Cell Death Detection Kit, POD (Roche, Mannheim, Germany). Nuclei were counterstained with 1.0 μg/mL 4′,6-diamidino-2-pheny-indole (DAPI; Beyotime, China) for 6 min to mark the nuclei and detected under a uorescence microscope (IX81; Olympus, Japan) after TUNEL reaction. Only TUNEL-positive cells that colocalized with DAPI-stained nuclei were counted as positive.

Immuno uorescence
The culture medium was exchanged with fresh medium, and the cells were incubated for 24 h and detected with a confocal laser scanning microscope (LSM 880 with Airyscan; Zeiss, Dublin, CA, USA). The nuclei were stained with DAPI for 15 min. The cells were imaged under a laser scanning confocal microscope (FV300; Olympus).

Animals
ApoE −/− mice with a C57BL/6 background were purchased from the Laboratory Animal Center of Peking University (Beijing, China). To verify the effect of TFEB on AS, male (aged 6 weeks) ApoE −/− mice were randomized into three groups of 10 (ND, HFD, and HFD+TFEB-shRNA-treated groups). The HFD mice were injected with lentivirus siRNA-TFEB (TFEB-shRNA) through the tail vein. Mice were fed an HFD (24% carbohydrate, 55% fat and 21% protein) for 16 weeks. Mice were anesthetized with inhalation of 2% sodium valproate, and 1.5 mL of blood was removed by cardiac puncture at week 16. The mice were euthanized by cervical dislocation, and tissues were collected for further analyses.

Statistical analysis
Data were analyzed using SPSS version 13.0 (SPSS, Chicago, IL, USA). Data are presented as the mean ± SD or median (interquartile range) unless otherwise indicated. The results were analyzed by one-way analysis of variance or unpaired Student's t-tests when continuous variables were normally distributed. A two-tailed P value <0.05 was considered statistically signi cant.

Declarations
Ethics approval and consent to participate All procedures with animals were approved by the Animal Care and Use Committee of Henan Provincial People' Hospital and were conducted following the institutional guidelines.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated/analyzed during the current study are available.

Competing interests
The authors declare that they have no con ict of interest.