NLRP3-inflammasome inhibition prevents high fat and high sugar diets-induced heart damage through autophagy induction

Oncotarget99740 www.impactjournals.com/oncotarget NLRP3-inflammasome inhibition prevents high fat and high sugar diets-induced heart damage through autophagy induction Luís E. Pavillard1,*, Diego Cañadas-Lozano1,*, Elísabet Alcocer-Gómez1, Fabiola Marín-Aguilar1, Sheila Pereira2, Avril A.B. Robertson3, Jordi Muntané4,7, Bernhard Ryffel5, Matthew A. Cooper3, José L. Quiles8, Pedro Bullón1,**, Jesús Ruiz-Cabello6,** and Mario D. Cordero8,** 1Research Laboratory, Oral Medicine Department, University of Sevilla, Sevilla, Spain 2Institute of Biomedicine of Seville (IBiS), “Virgen del Rocío” University Hospital, IBiS, CSIC, University of Seville, Seville, Spain 3Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia 4Department of General Surgery, Hospital Universitario Virgen del Rocio, CSIC, Universidad de Sevilla, Sevilla, Spain 5University and CNRS, UMR7355, Orléans, France 6CIBER de Enfermedades Respiratorias, Madrid, Spain; Advanced Imaging Unit, Centro Nacional de Investigaciones Cardiovasculares, and Universidad Complutense Madrid, Madrid, Spain 7Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD o Ciberehd), Instituto de Salud Carlos III, Madrid, Spain 8Institute of Nutrition and Food Technology “José Mataix Verdú”, Department of Physiology, Biomedical Research Center, University of Granada, Granada, Spain *These authors have contributed equally to this work **Co-senior author Correspondence to: Mario D. Cordero, email: [email protected] Keywords: NLRP3-inflammasome, cardiac damage, autophagy, MCC950 Received: June 28, 2017 Accepted: July 29, 2017 Published: September 08, 2017 Copyright: Pavillard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT The NLRP3-inflammasome complex has emerged as an important component of inflammatory processes in metabolic dysfunction induced by high-caloric diets. In this study, we investigate the molecular mechanisms by which NLRP3 inhibition may attenuate diet-induced cardiac injury. Here we show the cardiac damage induced by high sugar diet (HSD), high fat diet (HFD) or high sugar/fat diet (HSFD) over 15 weeks. Genetic ablation of NLRP3 protected against this damage by autophagy induction and apoptotic control. Furthermore, NLRP3 inhibition by the selective small molecule MCC950 resulted in similar autophagy induction and apoptotic control in hearts after diets. These data were reproduced in THP-1 cells treated with MCC950 and cultured in media supplemented with serum from mice dosed with MCC950 and fed with diets. NLRP3 inhibition exerted beneficial metabolic, and autophagic adaptations in hearts from obesogenic diets. The inhibition of NLRP3 activation may hold promise in the treatment of metabolic and cardiovascular diseases. INTRODUCTION Cardiovascular disease (CVD) is the leading cause of death in the world with a high prevalence both in industrialized but also in low- and middle-income countries [1]. Studies show that only about 8% US adults had a low risk profile for cardiovascular disease between 1999 and 2004 [2]. In Spain, the level of ideal www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 59), pp: 99740-99756 Research Paper Oncotarget99741 www.impactjournals.com/oncotarget cardiovascular health is as low as in the United States [3]. Energy-rich diets high in fat, processed and refined carbohydrate foods, together with sedentary lifestyles have led to an increased CVD risk, and this effect is thought to be mediated primarily by elevated LDL cholesterol blood levels. In these cases, the role of obesity is especially relevant because multifactorial pathophysiological mechanisms interrelate this condition with other diseases such as type 2 diabetes, renal disease, and metabolic syndrome. All these obesity-induced complications share a low-grade systemic inflammatory response characterized by upregulated cytokine production and activated signaling pathways [4], yet the specific inflammationrelated signaling pathways contributing to the metabolic dysfunction are still unknown. On this respect, the NLRP3-inflammasome complex has been described as a ‘danger sensor’, which triggers an innate immune response to a multitude of endogenous metabolic danger patterns inducing sterile inflammation [5]. NLRP3 activation induces the recruitment and autocatalytic activation of the cystein protease caspase-1, which processes the cytosolic precursors of the related cytokines IL-1β and IL-18, and leads to the secretion of the biologically active form of these cytokines [6]. Mice fed with HFD developed NLRP3- inflammasome activation in adipose, renal and hepatic tissues [7–9]. Interestingly, the absence of NLRP3 in these animals protected them to the adverse effects of HFD and the development of pathophysiology [7–9]. However, the role of NLRP3 in the pathogenesis of HFD- induced CVD appearance has not been characterized. Recent observations identified a specific role for NLRP3- inflammasome in the maladaptive response in cardiac tissue after ischemia/reperfusion in mice under high-fat high-fructose diets [10] and in the diabetic cardiomyopathy obseved after HFD and streptozotocin treatment in rats [11]. To understand the role of the NLRP3-inflammasome in diet induced-cardiac damage, we assesed the response of NLRP3 -/- mice, and the effect of the NLRP3 inhibitor MCC950 on high sugar diet (HSD), HFD and high sugar/ fat diet (HSFD). RESULTS Absence of the NLRP3 protects from HSD, HFD and HSFD-induced obesity We have investigated the role of the NLRP3- inflammasome in mice lacking the NLRP3 gene and exposed to the different diets inducing obesity. Food intake and bodyweight evolution were examined weekly during 15 weeks in each group. The different diets have been shown to induce significant weight gain in WT mice (Figures 1A and 1B) [7] despite increased food consumption that was similar in all WT and NLRP3- /- mice groups (Figures 1C and 1D). Predictably, hearts from WT mice were substantially heavier than SD-fed animals at the end of the study (p<0.05) (Figure 1E and Supplementary Figure 1). This finding was not altered in the NLRP3 -/- genotype on any obesity-associated diet. In humans, high-caloric diets cause numerous alterations including increased glucose, cholesterol levels and leptin/adiponectin dysregulation leading to cardiovascular disease, metabolic syndrome, and nonalcoholic fatty liver disease [12, 13]. The HSD, HFD and HSFD-fed WT mice showed increased serum levels of leptin (HSD and HFD, p<0.05; HSFD, p<0.001) and leptin/adiponectin ratio (p<0.001) (Figures 1F-1H), including abnormal serum levels of glucose, cholesterol, triglycerides, hepatic transaminases, uric acid and creatinine (Supplementary Table 2). These changes were not observed in NLRP3 -/- mice. NLRP3-deficiency induce a moderate protection against oxidative stress in heart and inflammation Oxidative stress has an essential role in inflammasome activation [14], and has been implicated in the cardiac pathophysiology induced by obesogenic diets [15]. Accordingly, analysis of oxidative stress biomarkers in hearts of HSD, HFD and HSFD fed WT mice revealed significant increases in lipid peroxidation (Figure 2A), protein carbonyls (Figure 2B) and of 8-oxoguanine glycosylase (OGG1) (Figures 2C and 2D). Compared to WT mice, NLRP3 -/- mice had a trend toward an elevated lipid peroxidation and OGG-1 levels (Figures 2A-2D). This finding was consistent with the antioxidant biomarkers were not increased in these knockout animals challenged with obesogenic diets, suggesting perhaps the absence of compensatory defense mechanisms (Figures 2E and 2F) in this genotype. These results were also accompanied by elevated serum inflammatory biomarkers such as IL-1β and TNF-α in WT mice (Figures 2G and 2H). Interestingly, no differences in serum levels of IL-1β and a moderate increase in TNF-α were observed in all NLRP3 -/- groups (Figures 2G and 2H). Because high fat diet is also associated to cardiomyocite apoptosis, which is coupled with oxidative stress and inflammation dysregulation [16], we determined several apoptotic biomarkers. Here HSD, HFD and HSFD feeding resulted in a significant increase in protein expression of cardiac BAX and active caspase 3 (Figures 3A, 3B and 3G) accompanied by reduced cardiac expression levels of antiapoptotic protein Bcl-2 (Figures 3C and 3G) in WT mice. NLRP3 -/- mice showed reduced contents of apoptosis-related markers, consistent with the accumulation of Bcl-2 which stimulates antiapoptotic activity in heart (Figures 3C and 3G). Interestingly, these observations were also accompanied by increased SIRT1 Oncotarget99742 www.impactjournals.com/oncotarget Figure 1: Nlrp3 signaling suppression prevents obesity induced effects of the HSD, HFD and HSFD diets. (A and B) Body weight chart of diets-induced obesity in WT and NLRP3 -/- mice. The mice were fed with the diets for 15 weeks and their body weights were monitored weekly. (C and D) Average daily oral food intake normalized to body weight, measured on the different diets. The food intake of mice was measured was monitored weekly. (E) Heart weight normalized to body weight. (F-H) Levels of leptin, adiponectin, and ratio in plasma. Blood samples were collected after overnight fasting. All data are presented as means ± SEM, n = 10 mice; *P < 0.05, *P < 0.005, ***P < 0.001. Oncotarget99743 www.impactjournals.com/oncotarget Figure 2: Nlrp3 signaling elimination protects against oxidative and inflammatory effects of the HSD, HFD and HSFD diets. (A and B) Levels of lipid peroxidation, and protein carbonyls in heart tissues from mice. (C) Protein expression level of 8-oxoguanine glycosylase (OGG-1), MnSOD and catalase. (D-F) Densitometric quantification of protein expression levels of western blots in heart tissues from mice relative to GAPDH (loading control). All data are presented as means ± SEM with a representative blot, n = 6–10 mice. (G and H) IL-1β and TNF-α levels in serum from WT and NLRP3 -/- mice fed with the differents diets were determined by ELISA. All data are presented as means ± SEM with a representative blot, n = 10 mice; *P < 0.05, ***P < 0.001 HSD, HFD and HSFD vs SD; aP < 0.005 WT vs NLRP3 -/-. Oncotarget99744 www.impactjournals.com/oncotarget Figure 3: Changes in apoptosis and autophagy in response to HSD, HFD and HSFD diets in WT and NLRP3 -/- mice. (A-C) Densitometric quantification of protein expression levels of Bax, Caspase 3 and Bcl-2 in heart tissues from mice relative to GAPDH (loading control). (D) Densitometric quantification of protein expression levels of Sirt1 in heart from mice. (E-F) Densitometric quantification of protein expression levels of autophagy markers in the heart. (G) Western blot analysis with representative blot including LC3. (H and I) Expression levels of Parkin and p62 level in the hearts of WT and NLRP3 -/- mice. All data are presented as means ± SEM, n =6–8 mice; * P < 0.05, ** P < 0.005, *** P < 0.001 HSD, HFD and HSFD vs SD; aP < 0.005 WT vs NLRP3 -/-. Oncotarget99745 www.impactjournals.com/oncotarget protein expression levels in knockout mice (Figures 3D and 3G) which has a role in heart protection, for example, by anti-inflammatory and antioxidant effects [16]. NLRP3-deficient mice show an autophagic protection against HSD, HFD and HSFD- Induced heart damage One of the most important mechanisms in the cardiac protection/injury is autophagy, which is impaired during aging and diet-induced obesity, producing a less effective clearance process and contributing to important cardiac damage [17]. HSD, HFD and HSFD induced high expressions of two important autophagic proteins such as Beclin 1 and ATG-12 (Figures 3E, 3F and 3G) in WT animals. Furthermore, we observed an impaired autophagic flux determined by expression of LC3-II and increased expression of proteins involved in clearance pathways such as PARKIN and p62/SQSTM1 (Figures 3G, 3H and 3I). Interestingly, all NLRP3 -/- groups showed an increased and more efficient autophagic machinery (Figures 3E-3I). As LC3-II overexpression can evoke either an increment of autophagy or an impairment of the autophagic flux, these LC3-II changes must to be interpreted together with the p62/SQSTM1 levels and minding that LC3-II itself is subject to autophagic degradation at the lysosome [18]. As, NLRP3 -/- mice showed a more efficient autophagy induction, as the lower levels of LC3-II (Figure 3G) and the reduced levels of proteins such as parkin (Figure 3H) and p62/SQSTM1 (Figure 3I) is suggesting. Changes in gene expression of adhesion molecules and fibrosis markers in heart To assess the impact of the diets on myocardial histology, cardiomyocyte cross-sectional area and fibrosis were examined. In the hematoxylin-and-eosin– stained sections, HSD and HFD but not HSFD showed a disarrangement of myofibrils and increased cardiomyocyte transverse cross-sectional area. However, NLRP3 -/- groups only showed a moderate increase in HFD (Figures 4A, 4B and Supplementary Figure 2). Further examination with Masson trichrome staining revealed overt perivascular fibrosis after diets in WT group without significant changes in NLRP3 -/- group (Figure 4A and Supplementary Figure 2). We also studied the changes in gene expression of several biomarkers of endothelial activation and fibrosis. Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were increased in heart tissues of HSD, HFD and HSFD-fed animals, but no significant changes were observed in NLRP3 -/- animals exposed to the same diets (Figure 4D). Similarly, typical fibrosis biomarkers such as Matrix metallopeptidase 2 (MMP2) and fibronectin, were also increased in heart tissues of HSD, HFD and HSFD-fed WT animals but again, nonoteworthy changes were observed in NLRP3 -/- mice (Figure 4D). In the same line, apoptosis biomarker caspase 3 and endotelial inflammatory biomarker COX-2 were also increased in heart tissues of HSD, HFD and HSFD-fed animals but again, whilst no significative changes were observed in knockout animals (Figure 4D). Pharmacological inhibition of NLRP3 prevent of the HSD, HFD and HSFD-Induced obesity and heart damage Using a pharmacological approximation, we have tested the effect of the same diets after inhibition of NLRP3 with MCC950. WT groups for this experiment were distributed in vehicle and the different diet+MCC950 (20 mg/Kg/day i.p.) animals. After fifteen weeks, all obesogenic diets led to increased weight in the vehicle group (Figure 5A), however the pharmacological inhibition of NLRP3- protected these animals from obesity (Figure 5A), similar to the genetic NLRP3 deletion. The daily food intake was lower in MCC950-treated groups compared to vehicle groups but, interestingly, with comparable relative changes among the different diets (Figure 5B). The different diets were shown to increase the heart weight in vehicle groups (Figure 5C and Supplementary Figure 3), with no change after MCC950 administration. Furthermore, MCC950 reduced cardiomyocytes area and perivascular fibrosis induced by diets (Supplementary Figure 4). The metabolic studies performed with these groups of animal showed how the treatment with the MCC950 inhibitor ameliorated the effect on several circulating metabolites. Thus, the increased leptin and leptin/ adiponectin ratio observed after HSD, HFD and HSFD feeding in vehicle groups, was reduced in the MCC950- treated groups with elevated adiponectin levels (Figures 5D-5F). Similarly, some alterations of biomarker levels in serum including glucose, cholesterol, triglycerides, hepatic transaminases, uric acid and creatinine (Supplementary Table 3) were reverted in the MCC950-treated mice. Guided by these findings on several metabolic pathways, we examined the effect of the obesogenic diets on oxidative stress, apoptosis and autophagy in heart tissues and compared to the observations described with NLRP -/- mice. As expected, we observed that the inhibitor resulted in a significant reduction of lipid peroxidation compared to those in the HSD, HFD and HSFD-vehicle groups together with a reduction of MnSOD protein expression (Figures 6A-6C). Additionally, MCC950 also induced down-regulation of the active caspase 3 apoptotic protein (Figures 6B and 6D). We also observed the same protective mechanisms in cardiac tissues determined by the high overexpression levels of SIRT1 in heart tissues of MCC950 treated groups (Figures 6B and 6E). Consistent with the NLRP3 Oncotarget99746 www.impactjournals.com/oncotarget Figure 4: Histological analyses and gene expression changes of adhesion molecules and fibrosis markers in heart. (A) Representative hematoxylin-and-eosin–stained micrographs showing transverse sections of Left Ventricular (LV) myocardium, crosssectional area and representative Masson trichrome–stained micrographs showing perivascular sections of myocardium of HFD from WT and NLRP3 -/-. Complete diets see Supplementary figure 2. (B) Quantitative analysis of cardiomyocyte cross-sectional (transverse) area with measurements of ≈100 cardiomyocytes from 3 to 6 mice per group. *P < 0.05, **P < 0.005, ***P < 0.001 HSD, HFD and HSFD vs SD. (C) Relative gene expressions of ICAM-1 and VCAM-1 (mean±SEM) determined by quantitative PCR in heart from WT and NLRP3 -/-. Relative gene expressions of MMP-2, and fibronectyn and relative gene expressions of Caspase 3 and COX-2 determined by quantitative PCR in heart from WT and NLRP3 -/-. All data are presented as means ± SEM, n =6 mice; *P < 0.05, **P < 0.005, ***P < 0.001 HSD, HFD and HSFD vs SD. Oncotarget99747 www.impactjournals.com/oncotarget Figure 5: NLRP3 inhibition by MCC950 prevents obesity induced effects of the HSD, HFD and HSFD diets. (A) Body weight chart of diets-induced obesity in WT-vehicle and MCC950 mice fed with HSD, HFD and HSFD diets. The mice were fed with the diets for 15 weeks and their body weights were monitored weekly. We represent the changes of the final week respect to the first week. (B) Average daily oral food intake normalized to body weight, measured on the different diets. The food intake of mice was measured was monitored weekly. We represent the changes of the final week respect to the first week. (C) Heart weight normalized to body weight. (D-F) Levels of leptin, adiponectin, and ratio in plasma. Blood samples were collected after overnight fasting. All data are presented as means ± SEM, n = 10 mice; *P < 0.05, ***P < 0.005 HSD, HFD and HSFD vs SD; aP < 0.05, aaP < 0.005 MCC950 vs vehicle. Oncotarget99748 www.impactjournals.com/oncotarget Figure 6: Changes in oxidative stress, apoptosis and autophagy in response to MCC950. (A) Lipid peroxidations. (B) Protein expression levels by western blot of MnSOD antioxidants in heart after MCC950 treatment in mice. We compare HSD, HFD and HSFD diets with MCC950 treated groups. (C) Densitometric quantification of protein expression levels of the antioxidant in heart tissues from mice relative to GAPDH (loading control). (D) Densitometric quantification of protein expression levels of the active Caspase 3 protein. (E) Densitometric quantification of protein expression levels of Sirt1 in heart from mice. 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