The E3 Ubiquitin Ligase Peli1 Deficiency Promotes Atherosclerosis Progression

Background: Atherosclerosis is a chronic inflammatory vascular disease and the main cause of death and morbidity. Emerging evidence suggests that ubiquitination plays an important role in the pathogenesis of atherosclerosis including control of vascular inflammation, vascular smooth muscle cell (VSMC) function and atherosclerotic plaque stability. Peli1 a type of E3 ubiquitin ligase has emerged as a critical regulator of innate and adaptive immunity, however, its role in atherosclerosis remains to be elucidated. Methods: Apoe−/− mice and Peli1-deficient Apoe−/− Peli1−/− mice were subject to high cholesterol diet. Post sacrifice, serum was collected, and atherosclerotic plaque size and parameters of atherosclerotic plaque stability were evaluated. Immunoprofiling and foam cell quantification were performed. Results: Peli1 deficiency does not affect atherosclerosis lesion burden and cholesterol levels, but promotes VSMCs foam cells formation, necrotic core expansion, collagen, and fibrous cap reduction. Apoe−/− Peli1−/− mice exhibit a storm of inflammatory cytokines, expansion of Th1, Th1, Th17, and Tfh cells, a decrease in regulatory T and B cells and induction of pro-atherogenic serum level of IgG2a and IgE. Conclusions: In the present study, we uncover a crucial role for Peli1 in atherosclerosis as an important regulator of inflammation and VSMCs phenotypic modulation and subsequently atherosclerotic plaque destabilization.


Introduction
Atherosclerosis is a chronic immunometabolic vascular disease and a main cause of death in both developed and developing countries [1,2]. The fibrous cap is an atheroprotective layer of vascular smooth muscle cells (VSMCs) that covers the atherosclerotic plaque [3] and which rupture induces acute thrombo-occlusive events, such as myocardial infarction and stroke [4]. Immune cells and inflammation play a key role in promoting the disruption of the fibrous cap [3]. Amongst the many pathophysiological factors, are also abnormal cholesterol metabolism, endothelial dysfunction, and VSMCs phenotypic modulation, and systemic inflammation all contributing to the progression of atherosclerosis however some of the precise molecular mechanisms underlying the development of atherosclerosis are not fully understood. Ubiquitination is a multiple-step process of post-translational protein modification involved in the regulation of many important cellular processes. Emerging evidence suggests that ubiquitination could play important roles in the pathogenesis of atherosclerosis particularly interfering with the regulation of vascular inflammation, and endothelial and VSMCs cell function which could altogether have a major impact on atherosclerotic plaque stability [5]. In the present study, we investigate the role of Peli1, a member of the Peli (Pellino) family of E3 ubiquitin ligases in atherosclerosis pathology.
Peli1 has been found to be involved in the regulation of the toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in innate immune cells [6][7][8]. In addition, Peli1 has been shown to modulate T cell receptor (TCR) signaling in T cells [8] and via control of TLR-mediated TRAF3 degradation and MAPK activation to trigger microglial activation and autoimmune inflammatory response [9]. Peli1 was also shown to be a critical regulator of the T cells activation as well as maintenance of peripheral T-cell tolerance [10]. Interestingly, Peli1 has been found to have a role in B cell autoantibody production in systemic lupus erythematosus pathogenesis, where via serving as an E3 ubiquitin ligase of NIK, it controlled Lys48-linked ubiquitination of NIK and subsequently noncanonical NF-κB activation [11]. Interestingly a recent publication has shown that Peli1 deficiency impairs the induction of the interleukin-1β (IL-1β) secretion by different NLRP3 triggers and it is required for NLRP3-induced caspase-1 activation and IL-1β maturation [12]. Peli1 conjugation to K63 ubiquitin chain to lysine 55 of the inflammasome adaptor apoptosisassociated speck-like protein containing (ASC), which in turn facilitates ASC/NLRP3 interaction and ASC oligomerization and subsequent inflammasome activation [12]. Since all the above-mentioned inflammatory signaling pathways particularly TLR, NF-κB, and NLRP3 activation have been shown by us and others to be major drivers of atherosclerosis disease progression and atherosclerosis pathology [13][14][15][16] on one hand and considering the involvement of Peli1 in their regulation, we investigated how Peli1 deficiency in an advanced model of atherosclerosis affects atherosclerosis disease progression.
Interestingly, the present study revealed that Peli1 plays a role of a critical regulator of systemic and local vascular inflammation, VSMCs foam cells formation which altogether impacts atherosclerotic plaque stability.

Mice
All mice were on a C57BL/6 background. Apoe −/− mice were first crossed with Peli1 −/− mice and the resulting F1 mice were then backcrossed on the Apoe −/− background. Eleven-week-old male Apoe −/− C57Bl/6 and Apoe −/− Peli1 −/− mice (crossed for 5 generations) were fed a high cholesterol diet (HCD) for 11 weeks (20.1% fat, 1.25% cholesterol, Research Diets, Inc., New Brunswick, NJ, USA) [17,18]. At sacrifice after overnight fasting, peripheral blood was collected by cardiac puncture. After the whole blood was clotted, it was centrifuged, and the serum was collected and stored at −80 • C until used. The systemic levels of cholesterol and triglyceride, nonessential fatty acids (NEFA), HDL (high-density lipoprotein), and glucose were evaluated using the in vitro tests for the quantitative determination LDL-Cholesterol Gen.3, HDL-Cholesterol Gen.4, TRIGL, and GLUC2, respectively (Roche Diagnostics GmbH, Mannheim, Germany). The measurement was performed using Cobas c 111 analyzers (Roche Diagnostics GmbH, Mannheim, Germany) at the University of Geneva Small Animal Phenotyping Core Facility. All experimental protocols and procedures were approved and performed according to the animal experimentation license (GE/100/16) issued by the Institutional Animal Care and Use Committee of the Geneva University School of Medicine. All procedures comply with the guidelines of Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and the NIH Guide for the Care and Use of Laboratory Animals.

Flow Cytometry
Initially, the mice were anaesthetized with 4% isoflurane (induction) followed by 2% isoflurane (maintenance) for peritoneal fluid collection. After checking the effectiveness of the anaesthesia, 5 mL of 2% BSA PBS was injected with a 10 mL syringe and a 20G needle into the peritoneal cavity of the mice. After a short massage of the abdomen, about 4 mL of liquid containing the peritoneal cells was collected with the same syringe. The procedure was performed a second time under the same anaesthesia to obtain enough liquid peritoneal (LP) mononuclear cells. The syringes and needles were changed between each mouse. Spleens (SP) and lymph nodes (LN) (inguinal, mesenterics, brachial, axillary, and superficial cervical) were smashed mechanically, and the obtained cell suspension was passed through a 70 µm cell strainer (BD Biosciences, MD, USA). Erythrocytes were lysed and nucleated cells were washed twice and counted. Lymphocytes were plated in U-bottom 96-well plate (1 × 10 6 cells/well) and stimulated in vitro with PMA (50 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) and ionomycin (Sigma-Aldrich, St. Louis, MO, USA), in the presence of brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) for 4 h. Before multi-color flow cytometry analysis to block nonspecific staining SP, LN or LP mononuclear cells (1 × 10 6 cells per sample) were incubated with anti-mouse FcRIIB/FcRIIIA mAb (BD Bioscience, Franklin Lakes, NJ, USA) and then stained with fluorophore-conjugated antibodies-CD4 AF488

Immunohistochemistry
Mouse aortic sinuses were serially cut at 5 µm as previously described [20,21]. The obtained sections were fixed in acetone and immunostained with specific anti-mouse MMP-9 antibody (R&D Systems, Minneapolis, MN, USA), anti-mouse CD68 (Serotec, Puchheim, Germany). All sections were counter-stained with Mayer's hemalum solution and rinsed in distilled water. Quantification was performed using the MetaMorph ® Microscopy Image Analysis Software from Molecular Devices or Definiens Developer 2.7 software (Definiens Inc., Carlsbad, CA, USA) for biomarker and morphological tissue profiling. The results were expressed as a stained area in lesion versus total lesion area.

Oil Red O Staining for Lipid Content
Five sections per mouse aortic roots and abdominal aorta were stained with Oil Red O, as previously described [20,21]. Aortas sections were counter-stained with Mayer's hemalum solution and rinsed in distilled water. Quantifications were performed using the Definiens Developer 2.7 software (Definiens Inc., Carlsbad, CA, USA). Data were calculated as ratios of Oil Red Oil positive area staining (lipid content) versus total lesion area. with ethanol for 30 s and cover-slipping, pictures of the sections were taken with ordinary polychromatic microscopy with identical exposure settings as previously described [22,23]. Quantifications were performed with Definiens Developer 2.7 software (Definiens Inc., Munich, Germany). Data were calculated as ratios of Sirius red positive stained positive area staining versus total lesion area.

Statistical Analysis
For comparison of two groups of continuous variables, two-tailed unpaired Mann-Whitney U-tests with a confidence level of 95% were conducted if data were non-normally distributed using GraphPad Prism 9. The number of mice used for each analysis is indicated in the figure legends. All data are presented as median with interquartile range with p ≤ 0.05 *, p ≤ 0.01 ** and p ≤ 0.001 ***.  Figure S1g). Peli1-deficient Apoe −/− mice fed HCD showed also a comparable with Apoe −/− mice atherosclerosis lesion size and lipid accumulation in the aortic roots and the abdominal aorta (Figure 1b-f). However, the percentage of aortic roots CD68 macrophages was significantly higher in Peli1-deficient Apoe −/− mice on HCD as quantified ( Figure 1d) and evident in the representative picture of CD68 staining in the aortic roots ( Figure 1g). Since high macrophages accumulation has been linked to plaque destabilization [24], we investigated the effect of Peli1-deficiency on markers of atherosclerotic plaque stability. Collagen plays a key role in atherosclerotic plaques stabilization and protection against rupture [25] and in comparison, to Apoe −/− mice, Apoe −/− Peli1 −/− mice fed HCD exhibited a significant reduction of collagen accumulation as quantified by Sirius red staining of aortic roots atherosclerotic lesions (Figure 1h

Peli1 Deficiency Affects Immune Cell Response in Atherosclerosis
Considering that inflammation in atherosclerosis is largely controlled by the adaptive immune system and in line with the previous findings showing that specific effect of Peli1 on peripheral T-cells, we observed that Peli1-deficiency promoted an increase in CD4 cells, Th1, Th2, T cells secreting IL-17 as well as T follicular helper cells (Figure 3a-e). In parallel the percentage of follicular regulatory T cells and Breg cells in the spleen were significantly reduced in Apoe −/− mice Peli1-deficient (Figure 3f

Peli1 Deficiency Affects Immune Cell Response in Atherosclerosis
Considering that inflammation in atherosclerosis is largely controlled by the adaptive immune system and in line with the previous findings showing that specific effect of Peli1 on peripheral T-cells, we observed that Peli1-deficiency promoted an increase in CD4 cells, Th1, Th2, T cells secreting IL-17 as well as T follicular helper cells (Figure 3a (Figure 6g-i). These results suggest that Peli1 abrogation induces global systemic inflammation. Additionally, the immunoglobulin IgE is shown to contribute to atherosclerosis and obesity by affecting macrophages polarization and foam cell formation [26] was also elevated systemically in Apoe −/− Peli1 −/− mice versus Apoe −/− mice in advanced atherosclerosis (Figure 6j). Peli1 has not only been linked to control of B cell autoantibody production, but Peli1-deficient mice showed elevated IgG2a [11], which is in line with our findings demonstrating that Peli1 deficient Apoe −/− mice on HCD have significantly elevated IgG2a levels.  (Figure 6g-i). These results suggest that Peli1 abrogation induces global systemic inflammation. Additionally, the immunoglobulin IgE is shown to contribute to atherosclerosis and obesity by affecting macrophages polarization and foam cell formation [26] was also elevated systemically in Apoe −/− Peli1 −/− mice versus Apoe −/− mice in advanced atherosclerosis (Figure 6j). Peli1 has not only been linked to control of B cell autoantibody production, but Peli1-deficient mice showed elevated IgG2a [11], which is in line with our findings demonstrating that Peli1 deficient Apoe −/− mice on HCD have significantly elevated IgG2a levels.

Discussion
The present study provides compelling evidence of the critical function of Peli1 E3 ubiquitin ligase in the pathogenesis of atherosclerosis. In advanced atherosclerosis, Peli1 deficiency appears to induce a multitude of detrimental effects. Although Peli1-abrogation did not affect atherosclerosis plaque size and cholesterol levels, Peli1 deficiency resulted in an elevation of the systemic inflammation including (1) induction of atherogenic immune cell populations-Th1 cells, CD4 L-17, and T follicular helper cells; (2) increased systemic pro-inflammatory cytokines like IL-6, TNF-a, IL-17, IL-18 and IL-12p70 as well as chemokines; (3) increased levels of circulating IgE and IgG2a. Moreover, Apoe −/− Peli-1 −/− mice exhibited several changes linked to atherosclerotic plaque destabilization like reduction of collagen accumulation and fibrous plaque thickness in parallel with an increased MMP9 expression and expansion of the necrotic core area. In parallel, Peli1 deficiency promoted VSMCs phenotypic modulation to macrophages like foam cells. Peli1 undoubtedly emerges as an important regulator of systemic and local vascular inflammation and which deficiency contributes to atherosclerosis progression linked to destabilization of the atherosclerotic plaques.
Inflammation has been proven to play a major role in all phases of atherosclerosis while ubiquitination could directly interfere with vascular inflammation, endothelial, and VSMCs cell function which could altogether affect important mechanisms of atherosclerosis disease development [5]. Peli1 has been found to be involved in the regulation of toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in innate immune cells [6][7][8] and modulating T cell receptor (TCR) signaling in T cells [8] and trigger an autoimmune inflammatory response. [9] Peli1 has been shown to be a critical regulator of the T cells activation as well as maintenance of peripheral T-cell tolerance [10]. Moreover, deficiency in a ubiquitin ligase, Peli1, causes hyperactivation of T cells and rendered T cells refractory to suppression by T regulatory cells and transforming growth factor (TGF)-β [27]. In line with this finding, we demonstrated in the present study that Peli1 is a critical factor in the maintenance of peripheral T-cell tolerance since we observed a pronounced increase of pro-atherogenic immune cells populations like Th1 subsets [28] and the proatherogenic Th17 and IL-17 producing cells [29] as well as Th2 cells and T follicular helper cells in Apoe −/− Peli1 −/− mice fed HCD. The frequency of T follicular helper subsets correlates positively with laboratory parameters of atherosclerosis progression suggesting the essential role of T follicular helper cells in promoting inflammatory response and atherosclerosis progression [30]. The induction of T cells in secondary lymphoid organs would subsequently prompt T cells to migrate to the atherosclerotic lesions where they could be reactivated and be involved in the modulation of the immunoinflammatory response locally in the atherosclerotic plaques [31], however, T cells recirculation to atherosclerotic lesions has not been demonstrated in the present study. Furthermore, peritoneal inflammatory cells have been shown to play a pivotal role in the development of experimental atherosclerosis [32]. In this regard, we observed that Peli1 deficiency triggers pro-atherogenic Th1 cells producing IFN-γ [33,34] as well as follicular helper T cells [30]. The observed systemic increase in pro-atherogenic T cells upon Peli1 abrogation highlight the important role of Peli1 in immune tolerance. Moreover, Peli1 deficiency was linked to a reduction of atheroprotective regulatory T and B cell subsets [35,36]. Peli1 has been demonstrated to be involved in the control of B cell autoantibody production in systemic lupus erythematosus via a negative regulator of the noncanonical NF-κB pathway in B cells resulting in the production of more antibodies, specifically promoting the IgG2a class-switch in Peli1-deficient mice [11]. In the present model of atherosclerosis, hypercholesteremia in Peli1-deficient mice reduced the percentage of FO B cells in the lymph nodes of Peli1 deficient mice, while in line with the previous findings the systemic level of IgG2a has been significantly elevated in Apoe −/− Peli1 −/− mice fed HCD. Moreover, IgG has been shown to aggravate atherosclerosis [37]. In parallel, we observed increased systemic level of the immunoglobulin IgE in Apoe −/− Peli1 −/− mice shown to contribute to atherosclerosis and foam cell formation [26]. In line with this finding, we found that Peli1 deficiency exacerbates VSMCs phenotypic switch to foam cells. After lipid engulfment via scavenger receptors, VSMCs become foam cells [38,39] and acquire expression of macrophage markers like-CD68, F4/80, and MAC2 [40] which altogether could promote the destabilization of atherosclerotic plaques. It has been demonstrated that about 30-70% of the VSMCs with lost contractile phenotype express macrophages markers. Macrophage-like VSMCs exhibit inflammatory properties and perform phagocytosis and recruit circulating inflammatory cells exacerbating the plaque inflammation. VSMC-derived foam cells compared with macrophages foam cells show impaired phagocytic capacity and reverse cholesterol transport [41]. Macrophage-like VSMCs have also been shown to release MMPs and promote neutrophil recruitment and increase plaque vulnerability [40,42]. Indeed, we observed that increased VSMCs foam cells formation occurs in parallel with the induction of parameters of characterizing vulnerable plaques. It has been demonstrated that active inflammation is linked to thinning of the fibrous cap, predisposing the plaque to rupture [43]. In this regard, it appears that although Peli1-deficiency does not affect the atherosclerotic plaque size it plays an important role in the control of the systemic inflammation and therefore in plaque structure stability and the regulation of MMP-9, collagen and necrotic core expansion, and fibrous cap formation as parameters known to have critical clinical importance for atherosclerotic plaque rupture.
The association between inflammation and atherosclerosis has been actively investigated in the last three decades and the role of inflammation in the pathogenesis and progression of atherogenesis has been well established [44]. Endothelial dysfunction, oxidative stress, VSMCs macrophage foam cells accumulation, toll-like receptor signaling, NLPR-3 inflammasome formation, and subsequent pro-inflammatory cytokine production, such as TNF-α, IL-1β, IL-6 are few of the mechanisms implicated in the atherogenic process. Moreover, there is evidence that anti-inflammatory biologic drugs, such as anti-TNF-α and anti-IL1β agents, can decelerate the atherogenic process [45]. Peli1 has been found to be involved in the regulation of toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in innate immune cells [6][7][8], while the present study showed that Peli1 deficiency in atherosclerosis promotes a storm of pro-inflammatory cytokines and chemokines which have been shown to not only direct leukocytes to the sites of inflammation during atherogenesis, but they also play a role in cell homeostasis and atherosclerosis plaque stability. Moreover, in line Peli1 abrogation triggers also an increase in CXCL10 which is strongly associated with a pronounced atheromatous formation [46].
The present study undoubtedly demonstrates that Peli1 abrogation in advanced atherosclerosis promotes atherosclerosis progression associated with induction of parameters characterizing destabilization of atherosclerotic plaques due to the lost control of the immune tolerance and increased in the systemic inflammatory cytokines and proatherogenic immunoglobulin.

Conclusions
Inflammation is crucial for atherosclerosis progression independently of hypercholesteremia and atherosclerotic plaque progression, but the mechanism and the factors regulating the inflammatory activation are incompletely understood. We demonstrate for the first time the role of the E3 ubiquitin ligase Peli1 as an important regulator for inflammatory-associated plaque destabilization in atherosclerosis.