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Sirt6-induced autophagy restricted TREM-1-mediated pyroptosis in ox-LDL-treated endothelial cells: relevance to prognostication of patients with acute myocardial infarction

Sirt6-induced autophagy restricted TREM-1-mediated pyroptosis in ox-LDL-treated endothelial... Inflammation mediated by myeloid cells trigger receptors 1 (TREM-1) is important for atherosclerosis development, while sirtuin 6 (Sirt6) levels decrease in atheroscleoritc plaque. Here we demonstrate that oxidatively modified low density lipoprotein (ox-LDL)-treated endothelial cells (ECs) exhibited increased TREM-1-mediated pyroptosis and decreased Sirt6-induced autophagy. We show that high sTREM-1 and low sSirt6 levels were independent predictors of boosted endothelial microparticles (EMPs) on admission, and were associated with increased risk for all-cause mortality and major adverse cardiovascular events (MACE) at median 24 months (interquartile range, 18–26) follow-up in acute myocardial infarction (AMI) patients. Additionally, blockage of Sirt6-induced autophagy led to augmented TREM-1- mediated pyroptosis, whereas Sirt6 overexpression attenuated ECs inflammation and pyroptosis following ox-LDL treatment. Our findings indicate that TREM-1 and in a reversed trend Sirt6 appeared to be markers of endothelial inflammation with potential for use in risk stratification. Introduction interleukin (IL)-6, IL-1β, and high sensitivity C-reactive Inflammation plays a critical role in atherosclerosis and protein were associated with an increased risk of no atherothrombosis beyond that of hypercholesteremia . ST-segment resolution and cardiovascular events in For instance, in the CANTOS trial, anti-inflammation patients undergoing emergent percutaneous coronary treatment with canakinumab led to a significantly lower intervention (PCI) , and that level of endothelial micro- rate of recurrent cardiovascular events independent of particles (EMPs), which reflects endothelial damage, was lipid level . Moreover, clinical and experimental data increased in patients with acute coronary syndrome support an important role of endothelial inflammation in (ACS) . Yet, to date, the mechanism underlying the the genesis and progression of atherosclerosis .We association between increasing degree of endothelial reported that biomarkers of inflammation such as inflammation in atherosclerosis and higher rates of car- diovascular events remains unclear. In atherosclerotic lesions, especially in advanced plaques, there is increased cell death and inflammation, Correspondence: Chen Fei (riverapt@126.com)or Liu Xue-Bo (liuxb70@hotmail.com) with the former augmenting the latter . Cell death is Department of Cardiology, Shanghai Tongji Hospital, Tongji University, traditionally ascribed to apoptosis and necrosis; however, Shanghai, China other forms of cell death have been identified, including These authors contributed equally: Ye Zi, Yao Yi-An Edited by E. Sayan © 2019 The Author(s) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Zi et al. Cell Death Discovery (2019) 5:88 Page 2 of 14 pyroptosis . Pyroptosis is defined as inflammatory cell ox-LDL treatment (Fig. 1c). In terms of expression death, which is dependent on caspase-1 activation and of inflammatory factors, as shown in Fig. 1d, TNF-α and pro-IL-1β, pro-IL-18 maturation , and that is triggered by IL-1β mRNA expression increased following ox-LDL both infectious and noninfectious diseases. Recently, treatment, whereas that of IL-10 decreased. pyroptosis was associated with nicotine-promoted ather- To assess whether TREM-1 participates in ox-LDL- 9 10 osclerosis and ox-LDL-induced macrophage death , induced pyroptosis, we then examined TREM-1 expres- sion in ECs. As shown in Fig. 2a, TREM-1 expression was suggesting a critical role of pyroptosis in atherosclerosis and rendering its inhibition a therapeutic target. Autop- much higher in ox-LDL or LPS group compared with LDL hagy, a “housekeeper” function for maintaining cellular group, which indicated that TREM-1 may play a key role homeostasis, may control cellular export and release of in ox-LDL-mediated EC pyroptosis. Then we used over- IL-1β and determine the modality of cell death pro- expression (TREM-1 KI, Adv TREM-1) or knockdown gression, such as apoptosis, necrosis, and senescence . method (TREM-1 KD, LV TREM-1) to regulate EC Although inflammation might induce autophagy sup- TREM-1 expression, we found that adv TREM-1 or LV pression and pyroptosis activation , the relationship TREM-1 significantly changed TREM-1 mRNA expres- between pyroptosis and autophagy remains controversial. sion (Fig. 2b). Interestingly, TREM-1 KI directly induced The triggering receptor expressed on myeloid cells-1 ECs pyroptosis and EMPs release, whereas TREM-1 KD (TREM-1), i.e., in neutrophils and monocytes/macrophages, nearly abolished ox-LDL-mediated ECs pyroptosis is considered an amplifier of the innate immune response in (Fig. 2c, d). Also, TREM-1 overexpression induced TNF-α infectious and noninfectious inflammation . Recently, and IL-1β mRNA expression, whereas it inhibited IL-10 TREM-1 was shown to be a biomarker for atherosclerosis expression. Additionally, TREM-1 KD nearly abolished and ACS , and evidence suggests that its inhibition using the ox-LDL-mediated effects on inflammatory factors in genetic or pharmacological methods may limit the devel- ECs (Fig. 2e). opment of experimental atherosclerosis . TREM-1 also is expressed on vascular smooth muscle cells (VSMCs) and its ox-LDL restricted Sirt6-induced autophagy in ECs activation promotes VSMCs inflammation, proliferation, To examine the change in Sirt6-induced autophagy in and migration . It remains unclear if TREM-1 is expressed ECs under normal and ox-LDL conditions, we used on endothelial cells (ECs) and if it is associated with mRFP-GFP-LC3 to stain for autophagosomes. As shown EC inflammation. Sirtuin 6 (Sirt6), a member of the evo- in Fig. 3a, ECs treated with ox-LDL contained less mRFP- lutionarily conserved nicotinamide adenine dinucleotide- GFP-LC3 vacuoles in the cytoplasm, indicating that ox- dependent histone deacetylases, has been found to be LDL inhibited EC autophagosome formation. Levels of downregulated and to induce autophagy in diabetes melli- the autophagic biomarkers decreased following ox-LDL tus and atherosclerotic plaque .Sirt6 deficiency exacer- treatment in the presence or absence of chloroquine bates endothelial senescence and chronic noninfective (Fig. 3b). Interestingly, Sirt6 expression also was down- inflammation . We hypothesized that Sirt6 downregulation regulated significantly in ECs treated with ox-LDL may be a risk factor for poor prognosis and a trigger for (Fig. 3b). upregulation of endothelial inflammation in ACS patients. To determine the role of Sirt6 on ox-LDL-mediated EC The present study therefore was aimed at investigating autophagic inhibition, we established wide-type Sirt6 whether TREM-1 upregulation and Sirt6 downregulation overexpression by means of recombinant adenovirus are associated with severe endothelial damage and poor (Sirt6 KI, Adv Sirt6) and Sirt6 knockdown by means of prognosis in ACS patients, and to assess whether Sirt6- recombinant lentivirus (Sirt6 KD, LV Sirt6), respectively induced autophagy may restrict TREM-1-guided ECs pyr- (Fig. 3c). Based on western blot and mRFP-GFP-LC3 optosis following ox-LDL treatment. staining results, we found that Sirt6 KI rescued autopha- gic biomarkers expression and autophagosome formation Results in ox-LDL-treated ECs, whereas Sirt6 KD nearly abolished TREM-1-mediated endothelial cell pyroptosis following autophagy efflux in untreated ECs (Fig. 3d, e). The latter ox-LDL treatment observations suggested that Sirt6 could relieve autophagy As previously reported, we used ox-LDL (50 µg/ml) to efflux in ox-LDL-treated ECs. induce EC damage; LDL (50 µg/ml) and LPS (1 µg/ml) were used in parallel as negative- and positive-control, TREM-1 mediated autophagy-inhibition in ox-LDL-treated respectively. As shown in Fig. 1a, three-color confocal ECs imaging revealed that ox-LDL significantly induced EC To evaluate whether ox-LDL-induced autophagy inhibi- pyroptosis. Two-color flow cytometry also showed that tion is associated with TREM-1 activation, we directly ox-LDL treatment induced a sixfold increase in pyroptotic regulated TREM-1 expression in ECs. As shown in Fig. 4a, EC number (Fig. 1b). EMP number increased following b, TREM-1 KI directly impeded autophagosome formation Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 3 of 14 Fig. 1 ox-LDL induces ECs pyroptosis. a Confocal detection of ox-LDL-induced pyroptotic cells formation. LDL was used as a negative control, and LPS as a positive control. Green: 488-labeled Casp1; red: TMR red-labeled pyroptotic nucleus; blue: DAPI-labeled natural nucleus; green and red double-labeled cells correspond to pyroptotic cells. N = 3. Scale bar: 50 μm. b Flow cytometry detecting ox-LDL-induced pyroptotic cells percentage. N = 3; *p < 0.05 versus LDL group. c. Flow cytometry detection of ox-LDL-induced EMPs number. N = 3; *p < 0.05 versus LDL group. d RT-PCR detection of mRNA expression of ox-LDL-induced inflammatory factors. N = 3; *p < 0.05 versus LDL group as with ox-LDL treatment, whereas TREM-1 KD rescued restrained EC pyroptosis following ox-LDL treatment or autophagic efflux in ECs. Also, we detected autophagic TREM-1 KI, whereas Sirt6 KD promoted ECs pyroptosis biomarkers expression following TREM-1 regulation. even in TREM-1 KD ECs (Fig. 5b–f). Also, induced Interestingly, TREM-1 KI decreased autophagic biomarkers expression of Sirt6 significantly decreased TNF-α and expression in ECs (Fig. 4c). Moreover, TREM-1 KD induced IL-1β mRNA expression, but increased IL-10 mRNA autophagic biomarkers re-expression in ox-LDL-treated expression in TREM-1 KI or ox-LDL-treated ECs ECs (Fig. 4d). (Fig. 5g). The latter experiments indicated that Sirt6 may reduce TREM-1-mediated pyroptosis in ox-LDL- Sirt6-induced autophagy restricted TREM-1-mediated treated ECs. pyroptosis in ox-LDL-treated ECs To further validate that the effect on TREM-1-mediated To determine the role of Sirt6-induced autophagy pyroptosis in ox-LDL-treated ECs is associated with Sirt6- in ECs pyroptosis, Sirt6 expression in TREM-1 KI ECs induced autophagy, we established autophagy-deficient was firstly detected. We found that TREM-1 KI sig- ECs using LV-shRNA of LC3 (Fig. 6a). Although LC3 KD nificantly downregulated Sirt6 expression (Fig. 5a). did not affect TREM-1 and Sirt6 expression (Fig. 6a), the Then, we evaluated the influenceofpyroptoticECs number of pyroptotic cells was much higher in LC3- on Sirt6-managed ECs. We found that Sirt6 KI deficient ECs (Fig. 6b–d) than LC3 intact ECs. Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 4 of 14 Fig. 2 TREM-1 participates in ox-LDL-induced ECs pyroptosis. a ox-LDL and LPS-induced TREM-1 expression. b Adv TREM-1 or LV TREM-1 significantly changed TREM-1 mRNA expression. c TREM-1 KI or TREM-1 KD effectively regulated ECs pyroptosis. N = 3. *p < 0.05 versus control group; p < 0.05 versus ox-LDL group. TREM-1 regulated pyroptotic cells formation (a) (Scale bar: 50 μm) and cell percentage (b), and Casp1 maturation. d TREM-1 regulated EMPs release following ox-LDL treatment. N = 3. *p < 0.05 versus control group; p < 0.05 versus ox-LDL group. e TREM-1 regulated inflammatory factors mRNA expression following ox-LDL treatment. N = 3. *p < 0.05 versus control group; p < 0.05 versus ox-LDL group Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 5 of 14 Fig. 3 Sirt6 participates in ox-LDL-decreased ECs autophagy. a ox-LDL-decreased ECs autophagosome formation. Green, GFP; red, RFP-labeled LC3; yellow, green, and yellow double-labeled autophagosome. Scale bar: 100 μm. b ox-LDL inhibited Sirt6 expression and decreased autophagic biomarkers expression with or without chloroquine. Chloro, Chloroquine. c Adv Sirt6 or LV Sirt6 significantly changed Sirt6 mRNA expression. d Sirt6 KI or Sirt6 KD effectively regulated ECs autophagic biomarkers expression. e Sirt6 KI or Sirt6 KD effectively regulated ECs autophagosome formation. N = 3. Scale bar: 100 μm sTREM-1, sSirt6, and clinical outcomes healthy controls (859.75 pg/ml, n = 68). Patients with Nine hundred and sixty-two STEMI and NSTEMI higher sTREM-1 (>median) or lower sSirt6 (<median) patients (median age, 62.7 years; 56.3% men) were were elderly and had a history of myocardial infarction enrolled in the study. The level of sTREM-1 was sig- and PCI or coronary artery bypass graft (CABG); a history nificantly higher in acute myocardial infarction (AMI) of smoking; nontreatment with ACEI/ARB or statin; and a patients (median 130.86 pg/ml) than in healthy control higher Killip class. (86.38 pg/ml, n = 68). In contrast, the levels of sSirt6 were At median 24 months (interquartile range, 18–26) lower in AMI patients (median 312.16 pg/ml) than in follow-up, the rate of all-cause mortality was 10.19% Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 6 of 14 Fig. 4 TREM-1 inhibits autophagy in ox-LDL-treated ECs. a TREM-1 KI directly inhibited autophagosome formation. Scale bar: 100 μm. b TREM-1 KD abrogated ox-LDL-mediated autophagosome formation inhibition. Scale bar: 100 μm. c TREM-1 KI directly inhibited autophagic biomarkers expression. d TREM-1 KD abrogated ox-LDL-mediated autophagic biomarkers expression inhibition. N = 3 (n = 98), and the rate of the combined major adverse We then divided patients into four categories according cardiovascular events (MACE) outcome was 18.71% (n = to high/low sTREM-1 and high/low sSirt6 using a cutoff at 180). Survival analysis showed that after adjustment for the median for sTREM-1 and sSirt6. The results based on variables such as age, male sex, smoking, hypertension, the Kaplan–Meier curves showed that patients with high diabetes, hypercholesterolemia, BMI, LVEF, and statin sTREM-1/low sSirt6 had the highest rate of all-cause use, the log sTREM-1 was a significant predictor of all- mortality (Fig. 7e) and MACE (Fig. 7f), whereas the patients cause mortality (HR: 1.61, 95% CI: 1.15–2.51; p < 0.01) with low sTREM-1/high sSirt6 had the lowest risk. and MACE (HR: 2.12, 95% CI: 1.26–3.55; p < 0.01), while the log sSirt6 was a negative predictor of all-cause mor- sTREM-1, sSirt6, and EMPs tality (HR: 0.73, 95% CI: 0.41–0.93; p < 0.01) but not of In this study, the level of EMPs was significantly higher MACE (HR: 0.86, 95% CI: 0.52–1.14; p = 0.42). in AMI patients (median 20,875/ml) than in healthy The log-rank test based on the Kaplan–Meier curves controls (median 10,218/ml); also the level of EMPs was also showed a significant association between high much higher in patients with all-cause mortality (median sTREM-1 and all-cause mortality (Fig. 7a) and MACE 29,000/µl) or MACE (median 24,615/µl). (Fig. 7b), and between low sSirt6 and all-cause mortality We also matched the levels of EMP and of sTREM-1/ (Fig. 7c) but not MACE (Fig. 7d). sSirt6. We found that sTREM-1 was related with EMP Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 7 of 14 Fig. 5 (See legend on next page.) Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 8 of 14 (see figure on previous page) Fig. 5 Sirt6 participates in TREM-1-mediated ECs pyroptosis. a TREM-1 inhibited Sirt6 expression. b TREM-1 and Sirt6 regulated Casp1 maturation. TREM-1 KI or Sirt6 KD directly induced Casp1 maturation, whereas Sirt6 KI effectively abolished ox-LDL- or TREM-1 KI-mediated Casp1 maturation; moreover, TREM-1 KD abrogated ox-LDL-mediated Casp1 maturation in Sirt6 intact ECs, but not in Sirt6 KD ECs. c Sirt6 KD simulated ox-LDL-induced pyroptotic cells formation, whereas Sirt6 KI prevented ox-LDL-induced pyroptotic cells formation. Scale bar: 50 μm. d TREM-1 KD abrogated ox-LDL- induced pyroptotic cells formation in Sirt6 intact ECs, but not in Sirt6 KD ECs. Scale bar: 50 μm. e Sirt6 KI stopped TREM-1 KI-induced pyroptotic cells formation. Scale bar: 50 μm. f Quantitation of pyroptotic cells using flow cytometry. g. TREM-1 and Sirt6 regulated inflammatory factors expression. TREM-1 KI or Sirt6 KD directly induced inflammatory factors expression, whereas Sirt6 KI effectively abolished ox-LDL- or TREM-1 KI-mediated inflammatory factors expression; moreover TREM-1 KD abrogated ox-LDL-mediated inflammatory factors expression in Sirt6 intact ECs, but not in # ## Sirt6 KD ECs. N = 3, *p < 0.05 versus control group; p < 0.05 versus ox-LDL group; **p < 0.05 versus TREM-1 KI group; p < 0.05 versus ox-LDL+ TREM-1 KD group increase, whereas sSirt6 was related with EMP decrease. By absence of cell death . Unlike Casp11, Casp1 does not using patients with low sTREM-1/high sSirt6 as the refer- have a canonical LPS binding domain, but it can stimulate ence group, EMP increase was significantly greater (HR: inflammasomes formation in microbe-associated mole- 6.08, 95% CI: 3.22–10.23; p < 0.01). In high sTREM-1/low cular patterns in infectious or noninfectious diseases . sSirt6 group, the level of EMPs reached 39000/µl, whereas Casp1-dependent pyroptosis plays key roles in limiting −/− the level of EMPs was 15,873/µl in low sTREM-1/high the spread of inflammation, Nlrc4 mice (which are sSirt6 group. Additionally, the levels of EMPs were statis- unable to normally activate Casp1) succumb to low tically similar in low sTREM-1/low sSirt6 group (20,615/µl) amounts of otherwise innocuous environmental oppor- and high sTREM-1/high sSirt6 group (18,598/µl). tunists . In atherosclerotic processing, Casp1-dependent pyroptosis activation seems to play a more critical role, Discussion such as in nicotine-mediated ECs dysfunction and ox- We conducted a series of in vitro experiments and LDL-induced form cell formation . The data presented in vivo assessments that led to the following findings: In here show that ox-LDL induces Casp1-dependent pyr- ox-LDL-treated ECs, Sirt6-induced autophagy restricted optosis activation in ECs; however, Casp11 expression is TREM-1-mediated pyroptosis, and in AMI patients, still nearly undetectable following ox-LDL treatment (data TREM-1 and in a reversed trend Sirt6 appeared to be not shown). Therefore, interventions focused on inflam- markers of endothelial inflammation with potential for mation and how to control phenotype switching of ECs in use in risk stratification. atherosclerosis might effectively prevent the onset or ECs are the first cells of the vascular wall to be damaged progression of the disease; however, the underlying in atherosclerosis triggered by ox-LDL, glucose, nicotine, mechanism remains unclear. and hypertension, and dysfunctional ECs play an impor- TREM-1 belongs to the immunoglobulin superfamily tant role in the development of atherosclerosis and ulti- and it is believed to play key roles in acute and chronic mately in inducing plaque rupture .ECinflammation and inflammatory disease. Inhibition of TREM-1 using phar- subsequent pyroptosis is considered the initiation and macological or genetic strategies significantly limits the critical step in this pathological process . Pyroptosis is a histological alterations and over-activation of host form of regulated cell death triggered by innate immunity, immune inflammatory responses in experimental mod- which manifests with specific morphological and mole- els . Of note, recent studies showed that TREM-1 may be cular features . A peculiar form of chromatin con- a reliable inflammatory mediator in atherosclerotic pro- densation without cellular swelling culminating as well as gression. Studies demonstrate that TREM-1 contributes in plasma membrane permeabilization, and IL-1 and to promoting monocytosis, monocyte/macrophage IL-18 release are the two main features of pyroptosis. proinflammatory responses , VSMCs inflammation, Initially, pyroptosis was only described as canonical Casp1 proliferation and migration , and formation of inflam- 19 15 activation in monocytes or macrophages . However matory foam cells . In clinical studies, sTREM-1 con- 26 27 recent findings indicate that pyroptosis also can be trig- centration is associated with diabetes and obesity , gered by several other caspases, such as Casp3, Casp11 which are risk factors for atherosclerosis. In long-term and is observed in other cell types, such as ECs follow-up shows that sTREM-1 concentration is not only and alveolar cells . Nevertheless, Casp11- or Casp1- associated with increased MACE and mortality in AMI dependent pyroptosis patterns are widely accepted and patients but also associated with in-stent restenosis in recognized. Casp11 has a highly specific physical binding ACS patients on statins . Additionally, genetic analysis domain of LPS, resulting in caspase oligomerization and revealed that TREM-1 gene polymorphisms are closely consequent activation. However, in some cell types associated with atherosclerosis severity in a Russian including DCs, Casp11 activation is observed in the population . Based on the latter evidence, one can posit Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 9 of 14 Fig. 6 LC3 KD induces pyroptosis. a LC3 KD did not change TREM-1 and Sirt6 expression. b LC3 KD directly provoked Casp1 maturation in ECs regardless of whether TREM-1 or Sirt6 was intact. c LC3 KD directly induced pyroptotic ECs formation regardless of whether TREM-1 or Sirt6 was intact. Scale bar: 50 μm. d Quantitation of pyroptotic cells using flow cytometry. N = 3, *p < 0.05 versus control group; p < 0.05 versus ox-LDL group; ## **p < 0.05 versus Sirt6 KI group; p < 0.05 versus ox-LDL+ TREM-1 KD group that elevated TREM-1 might, at least partially, reflect healthy controls. Interestingly, the increase in sTREM-1 endothelial inflammation in patients with atherosclerosis. was consistent with EMP level. However, previous Therefore, we assessed sTREM-1 and EMP levels in AMI immunofluorescence data showed that expression of patients, we found them to be significantly higher, espe- TREM-1 in CD45+ T or leukocytes sporadically localized 16,29 cially in the patients who suffered death or MACE, than in within the injured artery ; therefore, the possibility that Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 10 of 14 Fig. 7 sTREM-1 and sSirt6 levels on admission and 2-year clinical outcomes in 962 AMI patients. High sTREM-1 level was associated with increased risk for all-cause mortality (a) and MACE (b), while low sSirt6 level was associated with increased risk of all-cause mortality (c) but was not associated with MACE (d). e Patients with high sTREM-1/low sSirt6 had a higher rate of all-cause mortality, whereas those with low sTREM-1/high sSirt6 had a lower rate of all-cause mortality. f Patients with high sTREM-1/low sSirt6 had higher rate of MACE, whereas those with low sTREM-1/high sSirt6 had lower rate of MACE Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 11 of 14 EC inflammation is secondary to the activation of TREM- autophagy deficiency using 3-MA in acrolein-induced 1 and downstream pyroptosis cannot be excluded. For- Human umbilical vein endothelial cell (HUVECs) may tunately, we found that ox-LDL increased TREM-1 and reinforce pyroptosis, whereas, activating autophagy using Casp1 expression, and the proportion of pyroptotic cells rapamycin could inhibit pyroptosis . Much widely used among ECs. Direct induction of TREM-1-promoted cardiovascular drugs, such as Carvedilol, an α-, β-blocker expression of inflammatory factors, Casp1 activation and used to treat congestive heart failure and hypertension, EC pyroptosis, which were inhibited by TREM-1 shRNA. are being tested to attenuate macrophage pyroptosis and Thus, activation of TREM-1 on ECs seems to act as an induce autophagy activation in a Sirt6 dependent man- independent mechanism in promoting inflammatory cell ner . In this study, we demonstrated that direct LC3 death without other inflammatory cells. interference using shRNA reinforced Casp1 maturation Consistent with previous observations, TREM-1 and increased pyroptotic cell proportion. These data expression and activity are closely linked with both the underscore that autophagy restricts pyroptosis in many NOD-like receptors and Toll-like receptors [TLRs] , pathological conditions. It is worth noting that most which are involved in autophagy, a highly conserved previous studies focused on the role of NLRP3 and pro- multistep process aimed at maintaining cellular home- IL-1β, the major trigger of pyroptosis, on autophagy. They ostasis, including double membrane structure elongation confirmed that NLRP3 activation may suppress autophagy 35–37 (marked as p-AMPK, Atg5, and BECN1), autophagosome promotion , and autophagy may control pyroptosis formation (marked as LC3-II/LC3-I), fusion with lyso- progression through decrease IL-1β maturation from pro- some (marked as LAMP2), and finally cargo degeneration IL-1β and export . In our present study, TREM-1 KI (marked as p62) . Meanwhile, TLR activation induces directly induced an increase in pyroptotic cell proportion TREM-1 expression in a MyD88-dependent manner . and Casp1 maturation, but inhibited autophagic bio- Therefore, TREM-1 is suspected to regulate autophagy as markers expression and autophagosomes formation, suggested by previous studies. For example, TREM-like whileTREM-1 KD dampened pyroptosis but restored transcript-1-derived peptide [LR12] dampens TREM-1 autophagy in ox-LDL-treated HUVECs. Therefore, signaling, and TREM-1 genetic KO increases autophagic TREM-1 may also be a key regulator controlling the biomarkers, including TG1/ULK-1, Atg13, Atg5, pyroptosis/autophagy phenotype transition in ox-LDL- Atg16L1, LC3-I/II, HSPA8, and HSP90AA1 in colitic treated HUVECs. However, further molecular studies are mice. Further studies suggested that this effect may be needed to gain insight into the mechanisms underlying related with TREM-1-mediated mTOR downregulation the control by TREM-1 of the pyroptosis/autophagy and AMPK phosphorylation . Interestingly, TLR4 KO phenotype transition. may attenuate paraquat-elicited increase in LC3-II/LC3-I, Recently, several studies documented that cellular pyr- and phosphorylation of AMPK while decreasing phos- optosis/autophagy phenotype transition takes place in a 33 37 phorylation of mTOR in cardiomyocyte ; the effect of ROS-dependent manner . As a major scavenger of TREM-1 on autophagy thus remains controversial. In our endogenous ROS, Sirtuins are associated with anti- present study, we found that ox-LDL induced TREM-1 inflammation and pro-autophagy . Sirt6 is one of seven activation while impeding autophagy efflux in ECs. mammalian sirtuins, which plays an important role in cell Induced expression of TREM-1 directly inhibited autop- protection against various stress conditions in cardiovas- hagy efflux, including blunted autophagosome formation, cular diseases, and it is believed that its therapeutic effect decreased LC3-II/LC3-I, BECN1, LAMP2 expression, and may rely on autophagy initiation . Of note, Sirt6 defi- increased p62 expression. TREM-1 genetic KO using ciency triggers foam cell formation, endothelial senes- shRNA attenuates ox-LDL-elicited autophagy in ECs. cence and apoptosis, autophagy inhibition, and plaque TREM-1 thus might mediate autophagic abrogation in progression in atherosclerosis with or without diabetes . ox-LDL-treated ECs. Cells overexpressing Sirt6 are characterized by Although autophagy is believed to be inhibited in elevated autophagosome number, increased levels of inflammatory disease and the activation of autophagy may ATG proteins, such as ATG5 and LAMP2, and down- restrict inflammation in pathological or physiopathologi- regulation of the autophagic inhibition pathway, such cal progression, the accurate role of autophagy in pyr- as p53 and mTORC1 . Also, Sirt6 participates in cho- optosis is still controversial. For example, Atg7 deficiency lesterol metabolism in THP-1 cells . Our data show that intensifies inflammation and pyroptosis in Pseudomonas Sirt6 significantly alters number of autophagosomes and 34 35 sepsis and INF-mediated endometrial epithelial cells . the expression of autophagic biomarkers, such as LC3-II/ In contrast, autophagy deficiency using 3-MA in LC3-I, p62, and LAMP2, in ox-LDL-treated ECs, which benzoapyrene-induced HL-7702 cells may effectively indicates that Sirt6 induces autophagy and regulates dampen pyroptosis . Interestingly, autophagy restricts cholesterol metabolism in ECs. Sirt6 also protects cells by pyroptosis in cardiovascular diseases. For instance, suppressing the NF-kB and IL-1β signaling pathway in Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 12 of 14 inflammation, apoptosis and autophagy induction in both reagent and sTREM-1, the sSirt6 enzyme-linked immune 43,44 animals and humans and a feature of AS diseases . sorbent assay (ELISA) kit were purchased from Roche Also, IL-1β is the central factor of pyroptosis . Therefore, Applied Science (Indianapolis, USA). Monoclonal anti- Sirt6 is suspected to interfere with pyroptosis. Interest- bodies against CD62-PE, CD31-PE, CD42-FITC, and ingly, we found that ECs overexpressing Sirt6 are char- IgG-PE were purchased from BD (Shanghai, China), and acterized by decreased pyroptotic cell numbers, and TNF- monoclonal rabbit antibodies against Sirt6, TREM-1, LC3, BECN1, LAMP2, p62, Caspase-1, and GAPDH α and IL-1β mRNA levels, whereas Sirt6 deficiency aug- ments ox-LDL-mediated inflammation. The latter effects were purchased from Cell Signaling (Denver, Colorado, take place in an autophagy-dependent manner. Once USA). Fetal bovine serum (FBS) and Lipofectamine autophagy is blocked using LC3 shRNA, Sirt6-mediated 2000 Transfection Reagent were purchased from Invi- decreases of pyroptotic cells number and Casp-1 level are trogen (Carlsbad, CA, USA). The cDNA Synthesis Kit abrogated. Therefore, our identification of Sirt6-induced and Premix Ex Taq SYBR Green PCR Kit were purchased autophagy as a regulator of pyroptosis in ECs provides a from Takara (Shiga, Japan). The adv-Sirt6, LV-Sirt6 novel example of the multiple targets and functions of shRNA, adv-TREM-1, LV-TREM-1 shRNA, LV-LC3 Sirt6 in AS. However, Sirt6 involvement in TREM-1- shRNA, and GFR, GRP double labeled adv-LC3 were mediated pyroptosis and the underlying mechanisms have purchased from HanBio. (Shanghai, China). Other not been studied. Recently, Sirt6 was reported to be unmentioned reagents were purchased from Shenggong crucial in TLR4-, a TREM-1 activator , induced inflam- Bio. (Shanghai, China). mation. Sirt6 overexpression downregulates TLR4 and expression of inflammatory factors such as IL-1β, IL-6, Cell culture and TNF-α, whereas knocking down of Sirt6 activates HUVEC line was cultured in growth factor supple- TLR4 and inflammatory factors expression . In our mented endothelial cell media (ECM) from ScienCell present study, TREM-1 KI decreased Sirt6 expression, (Carlsbad, California, USA). After incubation with non- indicating that TREM-1-mediated proinflammatory effect FBS ECM for 6 h, HUVECs were reincubated in ECM might rely on Sirt6 abrogation. Sirt6 KI terminates with 5% FBS and LDL (100 ug/ml), ox-LDL (100 ug/ml) or TREM-1 KI or ox-LDL-induced Casp1 maturation, pyr- LPS (1 ug/ml) for 24 h. optotic cells formation and inflammatory factor secretion. We also used Sirt6 shRNA to produce Sirt6 KD cells. Sirt6 Adv and LV transfection KD directly induced pyroptosis activation regardless of Adv-control, LV-control, Adv-TREM-1, LV-TREM-1, TREM-1 intactness. These data indicate that TREM-1- Adv-Sirt6, LV-Sirt6, or LV-LC3B was directly added to mediated pyroptosis may be Sirt6 dependent, which cultured cells and incubated for 48 h. contrasts with the conclusion of a previous study . The cause of this difference may stem from differences in cell Analysis of cell pyroptosis lines or their handling. The complex association between Two-color flow cytometry and three-color confocal TREM-1 and Sirt6 warrants further study. imaging were used to detect cell pyroptosis. HUVECs In conclusion, the data presented here show that ox-LDL were incubated with Alexa Fluor 488 labeled caspase-1 at induced activation of pyroptosis occurs in a TREM-1 4 °C overnight, then stained with TMR red-labeled In-Situ dependent manner. Furthermore, TREM-1 negatively Cell Death Detection reagent according to manufacturer’s regulated the expression and activation of Sirt6. Moreover, protocol. Staining with DAPI was used for confocal the inhibitory effect of Sirt6 on inflammatory responses measurement. The caspase-1 and TMR double stained might be autophagy dependent. Additionally, in patients cells were considered as proptotic cells. with AMI, high levels of TREM-1, and low levels of Sirt6 were associated with an increased risk of all-cause mortality Analysis of cell autophagic efflux and MACE events at 2 years follow-up. The latter findings Two-color confocal imaging was used to detect cell suggest that TREM-1-mediated pyroptosis might underlie autophagic efflux. HUVECs were incubated with GFP the pro-atherosclerotic effect of ox-LDL, which may be labeled Adv-scramble RNA and GRP labeled Adv-LC3 for restricted by Sirt6-induced autophagy in ECs, thereby 48 h. Then the live cells were immediately examined by advancing our understanding of the pathophysiology of confocal imaging. The GFP and GRP double stained ox-LDL-induced ECs dysfunction in atherosclerosis. organelles were considered to be autophagic efflux. Methods Real-time reverse transcription PCR (RT-PCR) Reagents HUVECs were subjected to TRIzol to obtain total RNA TRIzol was purchased from Sigma (St Louis, Missouri, according to manufacturer’s instruction. Then, the total USA). TMR red-labeled In-Situ Cell Death Detection RNAs were converted to cDNA using Takara reverse Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 13 of 14 transcriptase and amplified using SYBR Premix reagent high-dose cardiac inotropes, diuretics, or intravenous for RT-PCR on an ABI 7500 system. The primers nitrate. (Shenggong Bio., China) were as follows: TNF-α Forward: 5’-CCGTCTCCTACCAGACCAAGG-3, Reverse: 5’-CT Blood samples GGAAGACCCCTCCCAGATAG-3’; IL-1β Forward: Blood samples were collected on admission and cen- 5’-CTGATGGCCCTAAACAGATGAAG-3’, Reverse: 5’- trifuged within 30 min to separate plasma which was GGTCGGAGATTCGTAGCAGCTGGAT-3’; IL-10 For- stored immediately at −80℃. The levels of sTREM-1 and ward: 5’-CATGCTGCTGGGCCTGAA-3’, Reverse: 5’-CG sSirt6 were measured using an established ELISA kit TCTCCTTGATCTGCTTGATG-3’. according to the guide manual. Western blotting Plasma EMPs collection and quantitative determination HUVECs were subjected to protein lysis buffer (Beyo- As previously described [5], after phlebotomy, the blood time, Haimen, china) to obtain total protein according to sample was buffered with sodium citrate immediately and manufacturer’s instruction. Equal amounts of total pro- centrifuged at 1,500 g for 10 min, and then, the super- tein were subjected to 8–12% SDS-PAGE, transferred to natant fluid was centrifuged at 13,000 g for 20 min, and PVDF membranes, blocked with 5% nonfat milk, incu- the pellet was resuspended in PBS to obtain EMPs con- bated with primary antibodies at 4 °C overnight, and then centration using flow cytometry. Because CD31 and CD62 coated with HRP-conjugated secondary antibodies at are biomarkers for ECs, and CD42 for platelets, CD62+/ room temperature for 1 h according to standardized CD42− or CD31+/CD42− were considered to be EMPs protocol. Finally, we used enhanced chemiluminescence in plasma. CD62+ EMPs are considered to be released reagents (Bio-Max, Israel) to visualize the immunoblots from activated ECs, whereas CD31+ EMPs are released reaction. from damaged ECs, therefore we detected the plasma levels of CD31+/CD42− EMPs. Clinical study population From October 2012 to December 2015, we pro- Statistical analysis spectively enrolled a total of 962 consecutive patients who Continuous variables are presented as median and presented with ST-segment or non-ST-segment elevation interquartile range and were compared by Mann–Whitney AMI to Shanghai Tongji Hospital, Shanghai East Hospital, U test. Categorical variables are presented as frequencies Tongji University, and Zhongshan Hospital, Fudan Uni- and were compared by chi-square test or Fisher’sExact versity. AMI was diagnosed according to the “Third uni- test. Log-rank tests and Cox proportional models were versal definition of myocardial infarction” . Individuals used to analyze the prognostic variables and clinical out- younger than 18 years and those with known acute or comes during follow-up and expressed as hazard ratios chronic infectious diseases, liver or renal replacement (HRs) with 95% confidence intervals. The factors entered therapy, malignancy, a suspected or known immuno- into the Cox proportional models were age, sex, hyper- compromised state, ongoing use or other systemic anti- tension, diabetes, smoking, hypercholesteremia, body mass inflammatory treatments and surgery in the previous index, MI history, PCI or CABG history, AMI type, Killip 1 month were excluded. Sixty-eight subjects without any class, peak troponin I, sTREM-1, sSirt6, and EMP level. signs of ACS and risk factors of CHD were used as healthy The C-statistic was used to evaluate the values of control group. All patients signed an informed consent sTREM-1, sSirt6, and EMP in the prediction of all-cause form and accepted management according to usual mortality and MACE events. Two-tailed unpaired Stu- practice. Blood samples were obtained immediately at the dent’s t-test or one-way analysis of variance tests were time of admission. used to determine the significance of the differences among the cellular experimental data. Values of p < 0.05 Clinical study outcomes were considered statistically significant. All data analyses All patients prospectively accepted to undergo a follow- were performed using SPSS 20.0 (SPSS Inc., Chicago, up at 2 years after presentation via telephone call to the USA). patients or their families and review of their medical Acknowledgments records. The data were entered into a central database and We thank Prof. Fei Zheng for helping us carry out the western blot verified by an authorized person. experiments. This work was supported by the China National Natural Science The clinical study outcomes were 2-year rates of all- Foundation, Nos: 81670403, 81500381, and 81370390; Shanghai science and Technology Commission medical guidance project, No: 18411950300; and the cause mortality and MACE including cardiovascular V.G. Youth Research Fund, No: 2017-CCA-VG-034. mortality and admission due to recurrent AMI, heart failure or unstable angina that led to urgent revascular- Conflict of interest ization. Heart failure was defined as that treated with The authors declare that they have no conflict of interest. Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 14 of 14 Publisher’s note 22. Lai, D. et al. Group 2 innate lymphoid cells protect lung endothelial cells from Springer Nature remains neutral with regard to jurisdictional claims in pyroptosis in sepsis. 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Trends Biochem. Sci. 42,245–254 (2017). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

Sirt6-induced autophagy restricted TREM-1-mediated pyroptosis in ox-LDL-treated endothelial cells: relevance to prognostication of patients with acute myocardial infarction

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Springer Journals
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Copyright © 2019 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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10.1038/s41420-019-0168-4
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Abstract

Inflammation mediated by myeloid cells trigger receptors 1 (TREM-1) is important for atherosclerosis development, while sirtuin 6 (Sirt6) levels decrease in atheroscleoritc plaque. Here we demonstrate that oxidatively modified low density lipoprotein (ox-LDL)-treated endothelial cells (ECs) exhibited increased TREM-1-mediated pyroptosis and decreased Sirt6-induced autophagy. We show that high sTREM-1 and low sSirt6 levels were independent predictors of boosted endothelial microparticles (EMPs) on admission, and were associated with increased risk for all-cause mortality and major adverse cardiovascular events (MACE) at median 24 months (interquartile range, 18–26) follow-up in acute myocardial infarction (AMI) patients. Additionally, blockage of Sirt6-induced autophagy led to augmented TREM-1- mediated pyroptosis, whereas Sirt6 overexpression attenuated ECs inflammation and pyroptosis following ox-LDL treatment. Our findings indicate that TREM-1 and in a reversed trend Sirt6 appeared to be markers of endothelial inflammation with potential for use in risk stratification. Introduction interleukin (IL)-6, IL-1β, and high sensitivity C-reactive Inflammation plays a critical role in atherosclerosis and protein were associated with an increased risk of no atherothrombosis beyond that of hypercholesteremia . ST-segment resolution and cardiovascular events in For instance, in the CANTOS trial, anti-inflammation patients undergoing emergent percutaneous coronary treatment with canakinumab led to a significantly lower intervention (PCI) , and that level of endothelial micro- rate of recurrent cardiovascular events independent of particles (EMPs), which reflects endothelial damage, was lipid level . Moreover, clinical and experimental data increased in patients with acute coronary syndrome support an important role of endothelial inflammation in (ACS) . Yet, to date, the mechanism underlying the the genesis and progression of atherosclerosis .We association between increasing degree of endothelial reported that biomarkers of inflammation such as inflammation in atherosclerosis and higher rates of car- diovascular events remains unclear. In atherosclerotic lesions, especially in advanced plaques, there is increased cell death and inflammation, Correspondence: Chen Fei (riverapt@126.com)or Liu Xue-Bo (liuxb70@hotmail.com) with the former augmenting the latter . Cell death is Department of Cardiology, Shanghai Tongji Hospital, Tongji University, traditionally ascribed to apoptosis and necrosis; however, Shanghai, China other forms of cell death have been identified, including These authors contributed equally: Ye Zi, Yao Yi-An Edited by E. Sayan © 2019 The Author(s) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Zi et al. Cell Death Discovery (2019) 5:88 Page 2 of 14 pyroptosis . Pyroptosis is defined as inflammatory cell ox-LDL treatment (Fig. 1c). In terms of expression death, which is dependent on caspase-1 activation and of inflammatory factors, as shown in Fig. 1d, TNF-α and pro-IL-1β, pro-IL-18 maturation , and that is triggered by IL-1β mRNA expression increased following ox-LDL both infectious and noninfectious diseases. Recently, treatment, whereas that of IL-10 decreased. pyroptosis was associated with nicotine-promoted ather- To assess whether TREM-1 participates in ox-LDL- 9 10 osclerosis and ox-LDL-induced macrophage death , induced pyroptosis, we then examined TREM-1 expres- sion in ECs. As shown in Fig. 2a, TREM-1 expression was suggesting a critical role of pyroptosis in atherosclerosis and rendering its inhibition a therapeutic target. Autop- much higher in ox-LDL or LPS group compared with LDL hagy, a “housekeeper” function for maintaining cellular group, which indicated that TREM-1 may play a key role homeostasis, may control cellular export and release of in ox-LDL-mediated EC pyroptosis. Then we used over- IL-1β and determine the modality of cell death pro- expression (TREM-1 KI, Adv TREM-1) or knockdown gression, such as apoptosis, necrosis, and senescence . method (TREM-1 KD, LV TREM-1) to regulate EC Although inflammation might induce autophagy sup- TREM-1 expression, we found that adv TREM-1 or LV pression and pyroptosis activation , the relationship TREM-1 significantly changed TREM-1 mRNA expres- between pyroptosis and autophagy remains controversial. sion (Fig. 2b). Interestingly, TREM-1 KI directly induced The triggering receptor expressed on myeloid cells-1 ECs pyroptosis and EMPs release, whereas TREM-1 KD (TREM-1), i.e., in neutrophils and monocytes/macrophages, nearly abolished ox-LDL-mediated ECs pyroptosis is considered an amplifier of the innate immune response in (Fig. 2c, d). Also, TREM-1 overexpression induced TNF-α infectious and noninfectious inflammation . Recently, and IL-1β mRNA expression, whereas it inhibited IL-10 TREM-1 was shown to be a biomarker for atherosclerosis expression. Additionally, TREM-1 KD nearly abolished and ACS , and evidence suggests that its inhibition using the ox-LDL-mediated effects on inflammatory factors in genetic or pharmacological methods may limit the devel- ECs (Fig. 2e). opment of experimental atherosclerosis . TREM-1 also is expressed on vascular smooth muscle cells (VSMCs) and its ox-LDL restricted Sirt6-induced autophagy in ECs activation promotes VSMCs inflammation, proliferation, To examine the change in Sirt6-induced autophagy in and migration . It remains unclear if TREM-1 is expressed ECs under normal and ox-LDL conditions, we used on endothelial cells (ECs) and if it is associated with mRFP-GFP-LC3 to stain for autophagosomes. As shown EC inflammation. Sirtuin 6 (Sirt6), a member of the evo- in Fig. 3a, ECs treated with ox-LDL contained less mRFP- lutionarily conserved nicotinamide adenine dinucleotide- GFP-LC3 vacuoles in the cytoplasm, indicating that ox- dependent histone deacetylases, has been found to be LDL inhibited EC autophagosome formation. Levels of downregulated and to induce autophagy in diabetes melli- the autophagic biomarkers decreased following ox-LDL tus and atherosclerotic plaque .Sirt6 deficiency exacer- treatment in the presence or absence of chloroquine bates endothelial senescence and chronic noninfective (Fig. 3b). Interestingly, Sirt6 expression also was down- inflammation . We hypothesized that Sirt6 downregulation regulated significantly in ECs treated with ox-LDL may be a risk factor for poor prognosis and a trigger for (Fig. 3b). upregulation of endothelial inflammation in ACS patients. To determine the role of Sirt6 on ox-LDL-mediated EC The present study therefore was aimed at investigating autophagic inhibition, we established wide-type Sirt6 whether TREM-1 upregulation and Sirt6 downregulation overexpression by means of recombinant adenovirus are associated with severe endothelial damage and poor (Sirt6 KI, Adv Sirt6) and Sirt6 knockdown by means of prognosis in ACS patients, and to assess whether Sirt6- recombinant lentivirus (Sirt6 KD, LV Sirt6), respectively induced autophagy may restrict TREM-1-guided ECs pyr- (Fig. 3c). Based on western blot and mRFP-GFP-LC3 optosis following ox-LDL treatment. staining results, we found that Sirt6 KI rescued autopha- gic biomarkers expression and autophagosome formation Results in ox-LDL-treated ECs, whereas Sirt6 KD nearly abolished TREM-1-mediated endothelial cell pyroptosis following autophagy efflux in untreated ECs (Fig. 3d, e). The latter ox-LDL treatment observations suggested that Sirt6 could relieve autophagy As previously reported, we used ox-LDL (50 µg/ml) to efflux in ox-LDL-treated ECs. induce EC damage; LDL (50 µg/ml) and LPS (1 µg/ml) were used in parallel as negative- and positive-control, TREM-1 mediated autophagy-inhibition in ox-LDL-treated respectively. As shown in Fig. 1a, three-color confocal ECs imaging revealed that ox-LDL significantly induced EC To evaluate whether ox-LDL-induced autophagy inhibi- pyroptosis. Two-color flow cytometry also showed that tion is associated with TREM-1 activation, we directly ox-LDL treatment induced a sixfold increase in pyroptotic regulated TREM-1 expression in ECs. As shown in Fig. 4a, EC number (Fig. 1b). EMP number increased following b, TREM-1 KI directly impeded autophagosome formation Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 3 of 14 Fig. 1 ox-LDL induces ECs pyroptosis. a Confocal detection of ox-LDL-induced pyroptotic cells formation. LDL was used as a negative control, and LPS as a positive control. Green: 488-labeled Casp1; red: TMR red-labeled pyroptotic nucleus; blue: DAPI-labeled natural nucleus; green and red double-labeled cells correspond to pyroptotic cells. N = 3. Scale bar: 50 μm. b Flow cytometry detecting ox-LDL-induced pyroptotic cells percentage. N = 3; *p < 0.05 versus LDL group. c. Flow cytometry detection of ox-LDL-induced EMPs number. N = 3; *p < 0.05 versus LDL group. d RT-PCR detection of mRNA expression of ox-LDL-induced inflammatory factors. N = 3; *p < 0.05 versus LDL group as with ox-LDL treatment, whereas TREM-1 KD rescued restrained EC pyroptosis following ox-LDL treatment or autophagic efflux in ECs. Also, we detected autophagic TREM-1 KI, whereas Sirt6 KD promoted ECs pyroptosis biomarkers expression following TREM-1 regulation. even in TREM-1 KD ECs (Fig. 5b–f). Also, induced Interestingly, TREM-1 KI decreased autophagic biomarkers expression of Sirt6 significantly decreased TNF-α and expression in ECs (Fig. 4c). Moreover, TREM-1 KD induced IL-1β mRNA expression, but increased IL-10 mRNA autophagic biomarkers re-expression in ox-LDL-treated expression in TREM-1 KI or ox-LDL-treated ECs ECs (Fig. 4d). (Fig. 5g). The latter experiments indicated that Sirt6 may reduce TREM-1-mediated pyroptosis in ox-LDL- Sirt6-induced autophagy restricted TREM-1-mediated treated ECs. pyroptosis in ox-LDL-treated ECs To further validate that the effect on TREM-1-mediated To determine the role of Sirt6-induced autophagy pyroptosis in ox-LDL-treated ECs is associated with Sirt6- in ECs pyroptosis, Sirt6 expression in TREM-1 KI ECs induced autophagy, we established autophagy-deficient was firstly detected. We found that TREM-1 KI sig- ECs using LV-shRNA of LC3 (Fig. 6a). Although LC3 KD nificantly downregulated Sirt6 expression (Fig. 5a). did not affect TREM-1 and Sirt6 expression (Fig. 6a), the Then, we evaluated the influenceofpyroptoticECs number of pyroptotic cells was much higher in LC3- on Sirt6-managed ECs. We found that Sirt6 KI deficient ECs (Fig. 6b–d) than LC3 intact ECs. Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 4 of 14 Fig. 2 TREM-1 participates in ox-LDL-induced ECs pyroptosis. a ox-LDL and LPS-induced TREM-1 expression. b Adv TREM-1 or LV TREM-1 significantly changed TREM-1 mRNA expression. c TREM-1 KI or TREM-1 KD effectively regulated ECs pyroptosis. N = 3. *p < 0.05 versus control group; p < 0.05 versus ox-LDL group. TREM-1 regulated pyroptotic cells formation (a) (Scale bar: 50 μm) and cell percentage (b), and Casp1 maturation. d TREM-1 regulated EMPs release following ox-LDL treatment. N = 3. *p < 0.05 versus control group; p < 0.05 versus ox-LDL group. e TREM-1 regulated inflammatory factors mRNA expression following ox-LDL treatment. N = 3. *p < 0.05 versus control group; p < 0.05 versus ox-LDL group Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 5 of 14 Fig. 3 Sirt6 participates in ox-LDL-decreased ECs autophagy. a ox-LDL-decreased ECs autophagosome formation. Green, GFP; red, RFP-labeled LC3; yellow, green, and yellow double-labeled autophagosome. Scale bar: 100 μm. b ox-LDL inhibited Sirt6 expression and decreased autophagic biomarkers expression with or without chloroquine. Chloro, Chloroquine. c Adv Sirt6 or LV Sirt6 significantly changed Sirt6 mRNA expression. d Sirt6 KI or Sirt6 KD effectively regulated ECs autophagic biomarkers expression. e Sirt6 KI or Sirt6 KD effectively regulated ECs autophagosome formation. N = 3. Scale bar: 100 μm sTREM-1, sSirt6, and clinical outcomes healthy controls (859.75 pg/ml, n = 68). Patients with Nine hundred and sixty-two STEMI and NSTEMI higher sTREM-1 (>median) or lower sSirt6 (<median) patients (median age, 62.7 years; 56.3% men) were were elderly and had a history of myocardial infarction enrolled in the study. The level of sTREM-1 was sig- and PCI or coronary artery bypass graft (CABG); a history nificantly higher in acute myocardial infarction (AMI) of smoking; nontreatment with ACEI/ARB or statin; and a patients (median 130.86 pg/ml) than in healthy control higher Killip class. (86.38 pg/ml, n = 68). In contrast, the levels of sSirt6 were At median 24 months (interquartile range, 18–26) lower in AMI patients (median 312.16 pg/ml) than in follow-up, the rate of all-cause mortality was 10.19% Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 6 of 14 Fig. 4 TREM-1 inhibits autophagy in ox-LDL-treated ECs. a TREM-1 KI directly inhibited autophagosome formation. Scale bar: 100 μm. b TREM-1 KD abrogated ox-LDL-mediated autophagosome formation inhibition. Scale bar: 100 μm. c TREM-1 KI directly inhibited autophagic biomarkers expression. d TREM-1 KD abrogated ox-LDL-mediated autophagic biomarkers expression inhibition. N = 3 (n = 98), and the rate of the combined major adverse We then divided patients into four categories according cardiovascular events (MACE) outcome was 18.71% (n = to high/low sTREM-1 and high/low sSirt6 using a cutoff at 180). Survival analysis showed that after adjustment for the median for sTREM-1 and sSirt6. The results based on variables such as age, male sex, smoking, hypertension, the Kaplan–Meier curves showed that patients with high diabetes, hypercholesterolemia, BMI, LVEF, and statin sTREM-1/low sSirt6 had the highest rate of all-cause use, the log sTREM-1 was a significant predictor of all- mortality (Fig. 7e) and MACE (Fig. 7f), whereas the patients cause mortality (HR: 1.61, 95% CI: 1.15–2.51; p < 0.01) with low sTREM-1/high sSirt6 had the lowest risk. and MACE (HR: 2.12, 95% CI: 1.26–3.55; p < 0.01), while the log sSirt6 was a negative predictor of all-cause mor- sTREM-1, sSirt6, and EMPs tality (HR: 0.73, 95% CI: 0.41–0.93; p < 0.01) but not of In this study, the level of EMPs was significantly higher MACE (HR: 0.86, 95% CI: 0.52–1.14; p = 0.42). in AMI patients (median 20,875/ml) than in healthy The log-rank test based on the Kaplan–Meier curves controls (median 10,218/ml); also the level of EMPs was also showed a significant association between high much higher in patients with all-cause mortality (median sTREM-1 and all-cause mortality (Fig. 7a) and MACE 29,000/µl) or MACE (median 24,615/µl). (Fig. 7b), and between low sSirt6 and all-cause mortality We also matched the levels of EMP and of sTREM-1/ (Fig. 7c) but not MACE (Fig. 7d). sSirt6. We found that sTREM-1 was related with EMP Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 7 of 14 Fig. 5 (See legend on next page.) Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 8 of 14 (see figure on previous page) Fig. 5 Sirt6 participates in TREM-1-mediated ECs pyroptosis. a TREM-1 inhibited Sirt6 expression. b TREM-1 and Sirt6 regulated Casp1 maturation. TREM-1 KI or Sirt6 KD directly induced Casp1 maturation, whereas Sirt6 KI effectively abolished ox-LDL- or TREM-1 KI-mediated Casp1 maturation; moreover, TREM-1 KD abrogated ox-LDL-mediated Casp1 maturation in Sirt6 intact ECs, but not in Sirt6 KD ECs. c Sirt6 KD simulated ox-LDL-induced pyroptotic cells formation, whereas Sirt6 KI prevented ox-LDL-induced pyroptotic cells formation. Scale bar: 50 μm. d TREM-1 KD abrogated ox-LDL- induced pyroptotic cells formation in Sirt6 intact ECs, but not in Sirt6 KD ECs. Scale bar: 50 μm. e Sirt6 KI stopped TREM-1 KI-induced pyroptotic cells formation. Scale bar: 50 μm. f Quantitation of pyroptotic cells using flow cytometry. g. TREM-1 and Sirt6 regulated inflammatory factors expression. TREM-1 KI or Sirt6 KD directly induced inflammatory factors expression, whereas Sirt6 KI effectively abolished ox-LDL- or TREM-1 KI-mediated inflammatory factors expression; moreover TREM-1 KD abrogated ox-LDL-mediated inflammatory factors expression in Sirt6 intact ECs, but not in # ## Sirt6 KD ECs. N = 3, *p < 0.05 versus control group; p < 0.05 versus ox-LDL group; **p < 0.05 versus TREM-1 KI group; p < 0.05 versus ox-LDL+ TREM-1 KD group increase, whereas sSirt6 was related with EMP decrease. By absence of cell death . Unlike Casp11, Casp1 does not using patients with low sTREM-1/high sSirt6 as the refer- have a canonical LPS binding domain, but it can stimulate ence group, EMP increase was significantly greater (HR: inflammasomes formation in microbe-associated mole- 6.08, 95% CI: 3.22–10.23; p < 0.01). In high sTREM-1/low cular patterns in infectious or noninfectious diseases . sSirt6 group, the level of EMPs reached 39000/µl, whereas Casp1-dependent pyroptosis plays key roles in limiting −/− the level of EMPs was 15,873/µl in low sTREM-1/high the spread of inflammation, Nlrc4 mice (which are sSirt6 group. Additionally, the levels of EMPs were statis- unable to normally activate Casp1) succumb to low tically similar in low sTREM-1/low sSirt6 group (20,615/µl) amounts of otherwise innocuous environmental oppor- and high sTREM-1/high sSirt6 group (18,598/µl). tunists . In atherosclerotic processing, Casp1-dependent pyroptosis activation seems to play a more critical role, Discussion such as in nicotine-mediated ECs dysfunction and ox- We conducted a series of in vitro experiments and LDL-induced form cell formation . The data presented in vivo assessments that led to the following findings: In here show that ox-LDL induces Casp1-dependent pyr- ox-LDL-treated ECs, Sirt6-induced autophagy restricted optosis activation in ECs; however, Casp11 expression is TREM-1-mediated pyroptosis, and in AMI patients, still nearly undetectable following ox-LDL treatment (data TREM-1 and in a reversed trend Sirt6 appeared to be not shown). Therefore, interventions focused on inflam- markers of endothelial inflammation with potential for mation and how to control phenotype switching of ECs in use in risk stratification. atherosclerosis might effectively prevent the onset or ECs are the first cells of the vascular wall to be damaged progression of the disease; however, the underlying in atherosclerosis triggered by ox-LDL, glucose, nicotine, mechanism remains unclear. and hypertension, and dysfunctional ECs play an impor- TREM-1 belongs to the immunoglobulin superfamily tant role in the development of atherosclerosis and ulti- and it is believed to play key roles in acute and chronic mately in inducing plaque rupture .ECinflammation and inflammatory disease. Inhibition of TREM-1 using phar- subsequent pyroptosis is considered the initiation and macological or genetic strategies significantly limits the critical step in this pathological process . Pyroptosis is a histological alterations and over-activation of host form of regulated cell death triggered by innate immunity, immune inflammatory responses in experimental mod- which manifests with specific morphological and mole- els . Of note, recent studies showed that TREM-1 may be cular features . A peculiar form of chromatin con- a reliable inflammatory mediator in atherosclerotic pro- densation without cellular swelling culminating as well as gression. Studies demonstrate that TREM-1 contributes in plasma membrane permeabilization, and IL-1 and to promoting monocytosis, monocyte/macrophage IL-18 release are the two main features of pyroptosis. proinflammatory responses , VSMCs inflammation, Initially, pyroptosis was only described as canonical Casp1 proliferation and migration , and formation of inflam- 19 15 activation in monocytes or macrophages . However matory foam cells . In clinical studies, sTREM-1 con- 26 27 recent findings indicate that pyroptosis also can be trig- centration is associated with diabetes and obesity , gered by several other caspases, such as Casp3, Casp11 which are risk factors for atherosclerosis. In long-term and is observed in other cell types, such as ECs follow-up shows that sTREM-1 concentration is not only and alveolar cells . Nevertheless, Casp11- or Casp1- associated with increased MACE and mortality in AMI dependent pyroptosis patterns are widely accepted and patients but also associated with in-stent restenosis in recognized. Casp11 has a highly specific physical binding ACS patients on statins . Additionally, genetic analysis domain of LPS, resulting in caspase oligomerization and revealed that TREM-1 gene polymorphisms are closely consequent activation. However, in some cell types associated with atherosclerosis severity in a Russian including DCs, Casp11 activation is observed in the population . Based on the latter evidence, one can posit Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 9 of 14 Fig. 6 LC3 KD induces pyroptosis. a LC3 KD did not change TREM-1 and Sirt6 expression. b LC3 KD directly provoked Casp1 maturation in ECs regardless of whether TREM-1 or Sirt6 was intact. c LC3 KD directly induced pyroptotic ECs formation regardless of whether TREM-1 or Sirt6 was intact. Scale bar: 50 μm. d Quantitation of pyroptotic cells using flow cytometry. N = 3, *p < 0.05 versus control group; p < 0.05 versus ox-LDL group; ## **p < 0.05 versus Sirt6 KI group; p < 0.05 versus ox-LDL+ TREM-1 KD group that elevated TREM-1 might, at least partially, reflect healthy controls. Interestingly, the increase in sTREM-1 endothelial inflammation in patients with atherosclerosis. was consistent with EMP level. However, previous Therefore, we assessed sTREM-1 and EMP levels in AMI immunofluorescence data showed that expression of patients, we found them to be significantly higher, espe- TREM-1 in CD45+ T or leukocytes sporadically localized 16,29 cially in the patients who suffered death or MACE, than in within the injured artery ; therefore, the possibility that Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 10 of 14 Fig. 7 sTREM-1 and sSirt6 levels on admission and 2-year clinical outcomes in 962 AMI patients. High sTREM-1 level was associated with increased risk for all-cause mortality (a) and MACE (b), while low sSirt6 level was associated with increased risk of all-cause mortality (c) but was not associated with MACE (d). e Patients with high sTREM-1/low sSirt6 had a higher rate of all-cause mortality, whereas those with low sTREM-1/high sSirt6 had a lower rate of all-cause mortality. f Patients with high sTREM-1/low sSirt6 had higher rate of MACE, whereas those with low sTREM-1/high sSirt6 had lower rate of MACE Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 11 of 14 EC inflammation is secondary to the activation of TREM- autophagy deficiency using 3-MA in acrolein-induced 1 and downstream pyroptosis cannot be excluded. For- Human umbilical vein endothelial cell (HUVECs) may tunately, we found that ox-LDL increased TREM-1 and reinforce pyroptosis, whereas, activating autophagy using Casp1 expression, and the proportion of pyroptotic cells rapamycin could inhibit pyroptosis . Much widely used among ECs. Direct induction of TREM-1-promoted cardiovascular drugs, such as Carvedilol, an α-, β-blocker expression of inflammatory factors, Casp1 activation and used to treat congestive heart failure and hypertension, EC pyroptosis, which were inhibited by TREM-1 shRNA. are being tested to attenuate macrophage pyroptosis and Thus, activation of TREM-1 on ECs seems to act as an induce autophagy activation in a Sirt6 dependent man- independent mechanism in promoting inflammatory cell ner . In this study, we demonstrated that direct LC3 death without other inflammatory cells. interference using shRNA reinforced Casp1 maturation Consistent with previous observations, TREM-1 and increased pyroptotic cell proportion. These data expression and activity are closely linked with both the underscore that autophagy restricts pyroptosis in many NOD-like receptors and Toll-like receptors [TLRs] , pathological conditions. It is worth noting that most which are involved in autophagy, a highly conserved previous studies focused on the role of NLRP3 and pro- multistep process aimed at maintaining cellular home- IL-1β, the major trigger of pyroptosis, on autophagy. They ostasis, including double membrane structure elongation confirmed that NLRP3 activation may suppress autophagy 35–37 (marked as p-AMPK, Atg5, and BECN1), autophagosome promotion , and autophagy may control pyroptosis formation (marked as LC3-II/LC3-I), fusion with lyso- progression through decrease IL-1β maturation from pro- some (marked as LAMP2), and finally cargo degeneration IL-1β and export . In our present study, TREM-1 KI (marked as p62) . Meanwhile, TLR activation induces directly induced an increase in pyroptotic cell proportion TREM-1 expression in a MyD88-dependent manner . and Casp1 maturation, but inhibited autophagic bio- Therefore, TREM-1 is suspected to regulate autophagy as markers expression and autophagosomes formation, suggested by previous studies. For example, TREM-like whileTREM-1 KD dampened pyroptosis but restored transcript-1-derived peptide [LR12] dampens TREM-1 autophagy in ox-LDL-treated HUVECs. Therefore, signaling, and TREM-1 genetic KO increases autophagic TREM-1 may also be a key regulator controlling the biomarkers, including TG1/ULK-1, Atg13, Atg5, pyroptosis/autophagy phenotype transition in ox-LDL- Atg16L1, LC3-I/II, HSPA8, and HSP90AA1 in colitic treated HUVECs. However, further molecular studies are mice. Further studies suggested that this effect may be needed to gain insight into the mechanisms underlying related with TREM-1-mediated mTOR downregulation the control by TREM-1 of the pyroptosis/autophagy and AMPK phosphorylation . Interestingly, TLR4 KO phenotype transition. may attenuate paraquat-elicited increase in LC3-II/LC3-I, Recently, several studies documented that cellular pyr- and phosphorylation of AMPK while decreasing phos- optosis/autophagy phenotype transition takes place in a 33 37 phorylation of mTOR in cardiomyocyte ; the effect of ROS-dependent manner . As a major scavenger of TREM-1 on autophagy thus remains controversial. In our endogenous ROS, Sirtuins are associated with anti- present study, we found that ox-LDL induced TREM-1 inflammation and pro-autophagy . Sirt6 is one of seven activation while impeding autophagy efflux in ECs. mammalian sirtuins, which plays an important role in cell Induced expression of TREM-1 directly inhibited autop- protection against various stress conditions in cardiovas- hagy efflux, including blunted autophagosome formation, cular diseases, and it is believed that its therapeutic effect decreased LC3-II/LC3-I, BECN1, LAMP2 expression, and may rely on autophagy initiation . Of note, Sirt6 defi- increased p62 expression. TREM-1 genetic KO using ciency triggers foam cell formation, endothelial senes- shRNA attenuates ox-LDL-elicited autophagy in ECs. cence and apoptosis, autophagy inhibition, and plaque TREM-1 thus might mediate autophagic abrogation in progression in atherosclerosis with or without diabetes . ox-LDL-treated ECs. Cells overexpressing Sirt6 are characterized by Although autophagy is believed to be inhibited in elevated autophagosome number, increased levels of inflammatory disease and the activation of autophagy may ATG proteins, such as ATG5 and LAMP2, and down- restrict inflammation in pathological or physiopathologi- regulation of the autophagic inhibition pathway, such cal progression, the accurate role of autophagy in pyr- as p53 and mTORC1 . Also, Sirt6 participates in cho- optosis is still controversial. For example, Atg7 deficiency lesterol metabolism in THP-1 cells . Our data show that intensifies inflammation and pyroptosis in Pseudomonas Sirt6 significantly alters number of autophagosomes and 34 35 sepsis and INF-mediated endometrial epithelial cells . the expression of autophagic biomarkers, such as LC3-II/ In contrast, autophagy deficiency using 3-MA in LC3-I, p62, and LAMP2, in ox-LDL-treated ECs, which benzoapyrene-induced HL-7702 cells may effectively indicates that Sirt6 induces autophagy and regulates dampen pyroptosis . Interestingly, autophagy restricts cholesterol metabolism in ECs. Sirt6 also protects cells by pyroptosis in cardiovascular diseases. For instance, suppressing the NF-kB and IL-1β signaling pathway in Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 12 of 14 inflammation, apoptosis and autophagy induction in both reagent and sTREM-1, the sSirt6 enzyme-linked immune 43,44 animals and humans and a feature of AS diseases . sorbent assay (ELISA) kit were purchased from Roche Also, IL-1β is the central factor of pyroptosis . Therefore, Applied Science (Indianapolis, USA). Monoclonal anti- Sirt6 is suspected to interfere with pyroptosis. Interest- bodies against CD62-PE, CD31-PE, CD42-FITC, and ingly, we found that ECs overexpressing Sirt6 are char- IgG-PE were purchased from BD (Shanghai, China), and acterized by decreased pyroptotic cell numbers, and TNF- monoclonal rabbit antibodies against Sirt6, TREM-1, LC3, BECN1, LAMP2, p62, Caspase-1, and GAPDH α and IL-1β mRNA levels, whereas Sirt6 deficiency aug- ments ox-LDL-mediated inflammation. The latter effects were purchased from Cell Signaling (Denver, Colorado, take place in an autophagy-dependent manner. Once USA). Fetal bovine serum (FBS) and Lipofectamine autophagy is blocked using LC3 shRNA, Sirt6-mediated 2000 Transfection Reagent were purchased from Invi- decreases of pyroptotic cells number and Casp-1 level are trogen (Carlsbad, CA, USA). The cDNA Synthesis Kit abrogated. Therefore, our identification of Sirt6-induced and Premix Ex Taq SYBR Green PCR Kit were purchased autophagy as a regulator of pyroptosis in ECs provides a from Takara (Shiga, Japan). The adv-Sirt6, LV-Sirt6 novel example of the multiple targets and functions of shRNA, adv-TREM-1, LV-TREM-1 shRNA, LV-LC3 Sirt6 in AS. However, Sirt6 involvement in TREM-1- shRNA, and GFR, GRP double labeled adv-LC3 were mediated pyroptosis and the underlying mechanisms have purchased from HanBio. (Shanghai, China). Other not been studied. Recently, Sirt6 was reported to be unmentioned reagents were purchased from Shenggong crucial in TLR4-, a TREM-1 activator , induced inflam- Bio. (Shanghai, China). mation. Sirt6 overexpression downregulates TLR4 and expression of inflammatory factors such as IL-1β, IL-6, Cell culture and TNF-α, whereas knocking down of Sirt6 activates HUVEC line was cultured in growth factor supple- TLR4 and inflammatory factors expression . In our mented endothelial cell media (ECM) from ScienCell present study, TREM-1 KI decreased Sirt6 expression, (Carlsbad, California, USA). After incubation with non- indicating that TREM-1-mediated proinflammatory effect FBS ECM for 6 h, HUVECs were reincubated in ECM might rely on Sirt6 abrogation. Sirt6 KI terminates with 5% FBS and LDL (100 ug/ml), ox-LDL (100 ug/ml) or TREM-1 KI or ox-LDL-induced Casp1 maturation, pyr- LPS (1 ug/ml) for 24 h. optotic cells formation and inflammatory factor secretion. We also used Sirt6 shRNA to produce Sirt6 KD cells. Sirt6 Adv and LV transfection KD directly induced pyroptosis activation regardless of Adv-control, LV-control, Adv-TREM-1, LV-TREM-1, TREM-1 intactness. These data indicate that TREM-1- Adv-Sirt6, LV-Sirt6, or LV-LC3B was directly added to mediated pyroptosis may be Sirt6 dependent, which cultured cells and incubated for 48 h. contrasts with the conclusion of a previous study . The cause of this difference may stem from differences in cell Analysis of cell pyroptosis lines or their handling. The complex association between Two-color flow cytometry and three-color confocal TREM-1 and Sirt6 warrants further study. imaging were used to detect cell pyroptosis. HUVECs In conclusion, the data presented here show that ox-LDL were incubated with Alexa Fluor 488 labeled caspase-1 at induced activation of pyroptosis occurs in a TREM-1 4 °C overnight, then stained with TMR red-labeled In-Situ dependent manner. Furthermore, TREM-1 negatively Cell Death Detection reagent according to manufacturer’s regulated the expression and activation of Sirt6. Moreover, protocol. Staining with DAPI was used for confocal the inhibitory effect of Sirt6 on inflammatory responses measurement. The caspase-1 and TMR double stained might be autophagy dependent. Additionally, in patients cells were considered as proptotic cells. with AMI, high levels of TREM-1, and low levels of Sirt6 were associated with an increased risk of all-cause mortality Analysis of cell autophagic efflux and MACE events at 2 years follow-up. The latter findings Two-color confocal imaging was used to detect cell suggest that TREM-1-mediated pyroptosis might underlie autophagic efflux. HUVECs were incubated with GFP the pro-atherosclerotic effect of ox-LDL, which may be labeled Adv-scramble RNA and GRP labeled Adv-LC3 for restricted by Sirt6-induced autophagy in ECs, thereby 48 h. Then the live cells were immediately examined by advancing our understanding of the pathophysiology of confocal imaging. The GFP and GRP double stained ox-LDL-induced ECs dysfunction in atherosclerosis. organelles were considered to be autophagic efflux. Methods Real-time reverse transcription PCR (RT-PCR) Reagents HUVECs were subjected to TRIzol to obtain total RNA TRIzol was purchased from Sigma (St Louis, Missouri, according to manufacturer’s instruction. Then, the total USA). TMR red-labeled In-Situ Cell Death Detection RNAs were converted to cDNA using Takara reverse Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 13 of 14 transcriptase and amplified using SYBR Premix reagent high-dose cardiac inotropes, diuretics, or intravenous for RT-PCR on an ABI 7500 system. The primers nitrate. (Shenggong Bio., China) were as follows: TNF-α Forward: 5’-CCGTCTCCTACCAGACCAAGG-3, Reverse: 5’-CT Blood samples GGAAGACCCCTCCCAGATAG-3’; IL-1β Forward: Blood samples were collected on admission and cen- 5’-CTGATGGCCCTAAACAGATGAAG-3’, Reverse: 5’- trifuged within 30 min to separate plasma which was GGTCGGAGATTCGTAGCAGCTGGAT-3’; IL-10 For- stored immediately at −80℃. The levels of sTREM-1 and ward: 5’-CATGCTGCTGGGCCTGAA-3’, Reverse: 5’-CG sSirt6 were measured using an established ELISA kit TCTCCTTGATCTGCTTGATG-3’. according to the guide manual. Western blotting Plasma EMPs collection and quantitative determination HUVECs were subjected to protein lysis buffer (Beyo- As previously described [5], after phlebotomy, the blood time, Haimen, china) to obtain total protein according to sample was buffered with sodium citrate immediately and manufacturer’s instruction. Equal amounts of total pro- centrifuged at 1,500 g for 10 min, and then, the super- tein were subjected to 8–12% SDS-PAGE, transferred to natant fluid was centrifuged at 13,000 g for 20 min, and PVDF membranes, blocked with 5% nonfat milk, incu- the pellet was resuspended in PBS to obtain EMPs con- bated with primary antibodies at 4 °C overnight, and then centration using flow cytometry. Because CD31 and CD62 coated with HRP-conjugated secondary antibodies at are biomarkers for ECs, and CD42 for platelets, CD62+/ room temperature for 1 h according to standardized CD42− or CD31+/CD42− were considered to be EMPs protocol. Finally, we used enhanced chemiluminescence in plasma. CD62+ EMPs are considered to be released reagents (Bio-Max, Israel) to visualize the immunoblots from activated ECs, whereas CD31+ EMPs are released reaction. from damaged ECs, therefore we detected the plasma levels of CD31+/CD42− EMPs. Clinical study population From October 2012 to December 2015, we pro- Statistical analysis spectively enrolled a total of 962 consecutive patients who Continuous variables are presented as median and presented with ST-segment or non-ST-segment elevation interquartile range and were compared by Mann–Whitney AMI to Shanghai Tongji Hospital, Shanghai East Hospital, U test. Categorical variables are presented as frequencies Tongji University, and Zhongshan Hospital, Fudan Uni- and were compared by chi-square test or Fisher’sExact versity. AMI was diagnosed according to the “Third uni- test. Log-rank tests and Cox proportional models were versal definition of myocardial infarction” . Individuals used to analyze the prognostic variables and clinical out- younger than 18 years and those with known acute or comes during follow-up and expressed as hazard ratios chronic infectious diseases, liver or renal replacement (HRs) with 95% confidence intervals. The factors entered therapy, malignancy, a suspected or known immuno- into the Cox proportional models were age, sex, hyper- compromised state, ongoing use or other systemic anti- tension, diabetes, smoking, hypercholesteremia, body mass inflammatory treatments and surgery in the previous index, MI history, PCI or CABG history, AMI type, Killip 1 month were excluded. Sixty-eight subjects without any class, peak troponin I, sTREM-1, sSirt6, and EMP level. signs of ACS and risk factors of CHD were used as healthy The C-statistic was used to evaluate the values of control group. All patients signed an informed consent sTREM-1, sSirt6, and EMP in the prediction of all-cause form and accepted management according to usual mortality and MACE events. Two-tailed unpaired Stu- practice. Blood samples were obtained immediately at the dent’s t-test or one-way analysis of variance tests were time of admission. used to determine the significance of the differences among the cellular experimental data. Values of p < 0.05 Clinical study outcomes were considered statistically significant. All data analyses All patients prospectively accepted to undergo a follow- were performed using SPSS 20.0 (SPSS Inc., Chicago, up at 2 years after presentation via telephone call to the USA). patients or their families and review of their medical Acknowledgments records. The data were entered into a central database and We thank Prof. Fei Zheng for helping us carry out the western blot verified by an authorized person. experiments. This work was supported by the China National Natural Science The clinical study outcomes were 2-year rates of all- Foundation, Nos: 81670403, 81500381, and 81370390; Shanghai science and Technology Commission medical guidance project, No: 18411950300; and the cause mortality and MACE including cardiovascular V.G. Youth Research Fund, No: 2017-CCA-VG-034. mortality and admission due to recurrent AMI, heart failure or unstable angina that led to urgent revascular- Conflict of interest ization. Heart failure was defined as that treated with The authors declare that they have no conflict of interest. Official journal of the Cell Death Differentiation Association Zi et al. Cell Death Discovery (2019) 5:88 Page 14 of 14 Publisher’s note 22. Lai, D. et al. Group 2 innate lymphoid cells protect lung endothelial cells from Springer Nature remains neutral with regard to jurisdictional claims in pyroptosis in sepsis. 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Trends Biochem. Sci. 42,245–254 (2017). Official journal of the Cell Death Differentiation Association

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Cell Death Discovery – Springer Journals

Published: Apr 12, 2019

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