Identification of a leucine-mediated threshold effect governing macrophage mTOR signalling and cardiovascular risk.

High protein intake is common in western societies and is often promoted as part of a healthy lifestyle; however, amino-acid-mediated mammalian target of rapamycin (mTOR) signalling in macrophages has been implicated in the pathogenesis of ischaemic cardiovascular disease. In a series of clinical studies on male and female participants ( NCT03946774 and NCT03994367 ) that involved graded amounts of protein ingestion together with detailed plasma amino acid analysis and human monocyte/macrophage experiments, we identify leucine as the key activator of mTOR signalling in macrophages. We describe a threshold effect of high protein intake and circulating leucine on monocytes/macrophages wherein only protein in excess of ∼25 g per meal induces mTOR activation and functional effects. By designing specific diets modified in protein and leucine content representative of the intake in the general population, we confirm this threshold effect in mouse models and find ingestion of protein in excess of ∼22% of dietary energy requirements drives atherosclerosis in male mice. These data demonstrate a mechanistic basis for the adverse impact of excessive dietary protein on cardiovascular risk.

Loss of Macrophage mTORC2 Drives Atherosclerosis via FoxO1 and IL-1ß Signaling.

BACKGROUND: The mTOR (mechanistic target of rapamycin) pathway is a complex signaling cascade that regulates cellular growth, proliferation, metabolism, and survival. Although activation of mTOR signaling has been linked to atherosclerosis, its direct role in lesion progression and in plaque macrophages remains poorly understood. We previously demonstrated that mTORC1 (mTOR complex 1) activation promotes atherogenesis through inhibition of autophagy and increased apoptosis in macrophages. METHODS: Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. RESULTS: In contrast to the atheroprotective effect seen with blockade of macrophage mTORC1, macrophage-specific mTORC2-deficient mice exhibit an atherogenic phenotype, with larger, more complex lesions and increased cell death. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 (forkhead box protein O1) transcription factor, leading to suppression of proinflammatory pathways, especially the inflammasome/IL (interleukin)-1ß response, a key mediator of vascular inflammation and atherosclerosis. In addition, administration of FoxO1 inhibitors efficiently rescued the proinflammatory response caused by mTORC2 deficiency both in vitro and in vivo. Interestingly, collective deletion of macrophage mTOR, which ablates mTORC1- and mTORC2-dependent pathways, leads to minimal change in plaque size or complexity, reflecting the balanced yet opposing roles of these signaling arms. CONCLUSIONS: Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.

Author Correction: High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy.

High protein diets are commonly utilized for weight loss, yet have been reported to raise cardiovascular risk. The mechanisms underlying this risk are unknown. Here, we show that dietary protein drives atherosclerosis and lesion complexity. Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mTOR signaling. This is causal in plaque progression as the effects of dietary protein are abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis induced by atherogenic lipids, a process that involves mTORC1-dependent inhibition of mitophagy, accumulation of dysfunctional mitochondria, and mitochondrial apoptosis. Using macrophage-specific mTORC1- and autophagy-deficient mice we confirm this amino acid-mTORC1-autophagy signaling axis in vivo. Our data provide the first insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies will be important to define the vascular effects of protein-based weight loss regimens.

MAP4K4 negatively regulates CD8 T cell-mediated antitumor and antiviral immunity.

During cytotoxic T cell activation, lymphocyte function-associated antigen-1 (LFA-1) engages its ligands on antigen-presenting cells (APCs) or target cells to enhance T cell priming or lytic activity. Inhibiting LFA-1 dampens T cell-dependent symptoms in inflammation, autoimmune diseases, and graft-versus-host disease. However, the therapeutic potential of augmenting LFA-1 function is less explored. Here, we show that genetic deletion or inhibition of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) enhances LFA-1 activation on CD8 T cells and improves their adherence to APCs or LFA-1 ligand. In addition, loss of Map4k4 increases CD8 T cell priming, which culminates in enhanced antigen-dependent activation, proliferation, cytokine production, and cytotoxic activity, resulting in impaired tumor growth and improved response to viral infection. LFA-1 inhibition reverses these phenotypes. The ERM (ezrin, radixin, and moesin) proteins reportedly regulate T cell-APC conjugation, but the molecular regulator and effector of ERM proteins in T cells have not been defined. In this study, we demonstrate that the ERM proteins serve as mediators between MAP4K4 and LFA-1. Last, systematic analyses of many organs revealed that inducible whole-body deletion of Map4k4 in adult animals is tolerated under homeostatic conditions. Our results uncover MAP4K4 as a potential target to augment antitumor and antiviral immunity.

A Clinically Applicable Gene-Expression Classifier Reveals Intrinsic and Extrinsic Contributions to Consensus Molecular Subtypes in Primary and Metastatic Colon Cancer.

PURPOSE: Four consensus molecular subtypes (CMS1-4) of colorectal cancer were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for colorectal cancer-related mortality, we examined the molecular and clinical attributes of CMS in metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: We developed a colorectal cancer-focused NanoString-based CMS classifier that is ideally suited to interrogate archival tissues. We successfully used this panel in the CMS classification of formalin-fixed paraffin-embedded (FFPE) tissues from mCRC cohorts, one of which is composed of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modeling of colorectal cancer in vivo at relevant sites. RESULTS: Using our classifier, we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in nonmetastatic early-stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared with other CMS. Assigning CMS to primary tumors and their matched metastases reveals mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs reveals differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic colorectal cancer. CONCLUSIONS: We describe the utility of a NanoString panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on colorectal cancer heterogeneity during disease progression.

TFEB and trehalose drive the macrophage autophagy-lysosome system to protect against atherosclerosis.

In the atherosclerotic plaque, macrophages are the key catabolic workhorse responsible for clearing lipid and dead cell debris. To survive the highly proinflammatory and lipotoxic plaque environment, macrophages must adopt strategies for maintaining tight homeostasis and self-renewal. Macroautophagy/autophagy is a pro-survival cellular pathway wherein damaged or excess cellular cargoes are encapsulated by a double-membrane compartment and delivered to the lysosome for hydrolysis. Previously, macrophage-specific autophagy deficiency has been shown to be atherogenic through several complementary mechanisms including hyperactivation of the inflammasome, defective efferocytosis, accumulation of cytotoxic protein aggregates, and impaired lipid degradation. Conversely, in a recent study we hypothesized that enhancing the macrophage autophagy-lysosomal system through genetic or pharmacological means could protect against atherosclerosis. We demonstrated that TFEB, a transcription factor master regulator of autophagy and lysosome biogenesis, coordinately enhances the function of this system to reduce atherosclerotic plaque burden. Further, we characterized the disaccharide trehalose as a novel inducer of TFEB with similar atheroprotective effects. Overall, these findings mechanistically interrogate the importance and therapeutic promise of a functional autophagy-lysosome degradation system in plaque macrophage biology.

N-3 PUFAs induce inflammatory tolerance by formation of KEAP1-containing SQSTM1/p62-bodies and activation of NFE2L2.

Inflammation is crucial in the defense against infections but must be tightly controlled to limit detrimental hyperactivation. Our diet influences inflammatory processes and omega-3 polyunsaturated fatty acids (n-3 PUFAs) have known anti-inflammatory effects. The balance of pro- and anti-inflammatory processes is coordinated by macrophages and macroautophagy/autophagy has recently emerged as a cellular process that dampens inflammation. Here we report that the n-3 PUFA docosahexaenoic acid (DHA) transiently induces cytosolic speckles of the autophagic receptor SQSTM1/p62 (sequestosome 1) (described as SQSTM1/p62-bodies) in macrophages. We suggest that the formation of SQSTM1/p62-bodies represents a fast mechanism of NFE2L2/Nrf2 (nuclear factor, erythroid 2 like 2) activation by recruitment of KEAP1 (kelch like ECH associated protein 1). Further, the autophagy receptor TAX1BP1 (Tax1 binding protein 1) and ubiquitin-editing enzyme TNFAIP3/A20 (TNF α induced protein 3) could be identified in DHA-induced SQSTM1/p62-bodies. Simultaneously, DHA strongly dampened the induction of pro-inflammatory genes including CXCL10 (C-X-C motif chemokine ligand 10) and we suggest that formation of SQSTM1/p62-bodies and activation of NFE2L2 leads to tolerance towards selective inflammatory stimuli. Finally, reduced CXCL10 levels were related to the improved clinical outcome in n-3 PUFA-supplemented heart-transplant patients and we propose CXCL10 as a robust marker for the clinical benefits mobilized by n-3 PUFA supplementation.

Exploiting macrophage autophagy-lysosomal biogenesis as a therapy for atherosclerosis.

Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy-lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1ß levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy-lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease.

Target acquired: Selective autophagy in cardiometabolic disease.

The accumulation of damaged or excess proteins and organelles is a defining feature of metabolic disease in nearly every tissue. Thus, a central challenge in maintaining metabolic homeostasis is the identification, sequestration, and degradation of these cellular components, including protein aggregates, mitochondria, peroxisomes, inflammasomes, and lipid droplets. A primary route through which this challenge is met is selective autophagy, the targeting of specific cellular cargo for autophagic compartmentalization and lysosomal degradation. In addition to its roles in degradation, selective autophagy is emerging as an integral component of inflammatory and metabolic signaling cascades. In this Review, we focus on emerging evidence and key questions about the role of selective autophagy in the cell biology and pathophysiology of metabolic diseases such as obesity, diabetes, atherosclerosis, and steatohepatitis. Essential players in these processes are the selective autophagy receptors, defined broadly as adapter proteins that both recognize cargo and target it to the autophagosome. Additional domains within these receptors may allow integration of information about autophagic flux with critical regulators of cellular metabolism and inflammation. Details regarding the precise receptors involved, such as p62 and NBR1, and their predominant interacting partners are just beginning to be defined. Overall, we anticipate that the continued study of selective autophagy will prove to be informative in understanding the pathogenesis of metabolic diseases and to provide previously unrecognized therapeutic targets.

Sequences within the C Terminus of the Metabotropic Glutamate Receptor 5 (mGluR5) Are Responsible for Inner Nuclear Membrane Localization.

Traditionally, G-protein-coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However, recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5 (mGluR5) is concentrated at the inner nuclear membrane (INM) where it mediates Ca2+ changes in the nucleoplasm by coupling with Gq/11 Here, we identified a region within the C-terminal domain (amino acids 852-876) that is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known nuclear localization signal motifs, they represent a new motif for INM trafficking. mGluR5 is also trafficked to the plasma membrane where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with chromatin. Thus, mGluR5 is perfectly positioned to regulate nucleoplasmic Ca2+in situ.

Anti-angiogenic Nanotherapy Inhibits Airway Remodeling and Hyper-responsiveness of Dust Mite Triggered Asthma in the Brown Norway Rat.

Although angiogenesis is a hallmark feature of asthmatic inflammatory responses, therapeutic anti-angiogenesis interventions have received little attention. Objective: Assess the effectiveness of anti-angiogenic Sn2 lipase-labile prodrugs delivered via αvß3-micellar nanotherapy to suppress microvascular expansion, bronchial remodeling, and airway hyper-responsiveness in Brown Norway rats exposed to serial house dust mite (HDM) inhalation challenges. Results: Anti-neovascular effectiveness of αvß3-mixed micelles incorporating docetaxel-prodrug (Dxtl-PD) or fumagillin-prodrug (Fum-PD) were shown to robustly suppress neovascular expansion (p<0.01) in the upper airways/bronchi of HDM rats using simultaneous 19F/1H MR neovascular imaging, which was corroborated by adjunctive fluorescent microscopy. Micelles without a drug payload (αvß3-No-Drug) served as a carrier-only control. Morphometric measurements of HDM rat airway size (perimeter) and vessel number at 21d revealed classic vascular expansion in control rats but less vascularity (p<0.001) after the anti-angiogenic nanotherapies. CD31 RNA expression independently corroborated the decrease in airway microvasculature. Methacholine (MCh) induced respiratory system resistance (Rrs) was high in the HDM rats receiving αvß3-No-Drug micelles while αvß3-Dxtl-PD or αvß3-Fum-PD micelles markedly and equivalently attenuated airway hyper-responsiveness and improved airway compliance. Total inflammatory BAL cells among HDM challenged rats did not differ with treatment, but αvß3+ macrophages/monocytes were significantly reduced by both nanotherapies (p<0.001), most notably by the αvß3-Dxtl-PD micelles. Additionally, αvß3-Dxtl-PD decreased BAL eosinophil and αvß3+ CD45+ leukocytes relative to αvß3-No-Drug micelles, whereas αvß3-Fum-PD micelles did not. Conclusion: These results demonstrate the potential of targeted anti-angiogenesis nanotherapy to ameliorate the inflammatory hallmarks of asthma in a clinically relevant rodent model.

Ursolic acid enhances macrophage autophagy and attenuates atherogenesis.

Macrophage autophagy has been shown to be protective against atherosclerosis. We previously discovered that ursolic acid (UA) promoted cancer cell autophagy. In the present study, we aimed to examine whether UA enhances macrophage autophagy in the context of atherogenesis. Cell culture study showed that UA enhanced autophagy of macrophages by increasing the expression of Atg5 and Atg16l1, which led to altered macrophage function. UA reduced pro-interleukin (IL)-1ß protein levels and mature IL-1ß secretion in macrophages in response to lipopolysaccharide (LPS), without reducing IL-1ß mRNA expression. Confocal microscopy showed that in LPS-treated macrophages, UA increased LC3 protein levels and LC3 appeared to colocalize with IL-1ß. In cholesterol-loaded macrophages, UA increased cholesterol efflux to apoAI, although it did not alter mRNA or protein levels of ABCA1 and ABCG1. Electron microscopy showed that UA induced lipophagy in acetylated LDL-loaded macrophages, which may result in increased cholesterol ester hydrolysis in autophagolysosomes and presentation of free cholesterol to the cell membrane. In LDLR(-/-) mice fed a Western diet to induce atherogenesis, UA treatment significantly reduced atherosclerotic lesion size, accompanied by increased macrophage autophagy. In conclusion, the data suggest that UA promotes macrophage autophagy and, thereby, suppresses IL-1ß secretion, promotes cholesterol efflux, and attenuates atherosclerosis in mice.

Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis.

Autophagy is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. Atherosclerotic plaque formation is enhanced in mice with macrophages deficient for the critical autophagy protein ATG5. We showed that exposure of macrophages to lipids that promote atherosclerosis increased the abundance of the autophagy chaperone p62 and that p62 colocalized with polyubiquitinated proteins in cytoplasmic inclusions, which are characterized by insoluble protein aggregates. ATG5-null macrophages developed further p62 accumulation at the sites of large cytoplasmic ubiquitin-positive inclusion bodies. Aortas from atherosclerotic mice and plaques from human endarterectomy samples showed increased abundance of p62 and polyubiquitinated proteins that colocalized with plaque macrophages, suggesting that p62-enriched protein aggregates were characteristic of atherosclerosis. The formation of the cytoplasmic inclusions depended on p62 because lipid-loaded p62-null macrophages accumulated polyubiquitinated proteins in a diffuse cytoplasmic pattern. Lipid-loaded p62-null macrophages also exhibited increased secretion of interleukin-1ß (IL-1ß) and had an increased tendency to undergo apoptosis, which depended on the p62 ubiquitin-binding domain and at least partly involved p62-mediated clearance of NLRP3 inflammasomes. Consistent with our in vitro observations, p62-deficient mice formed greater numbers of more complex atherosclerotic plaques, and p62 deficiency further increased atherosclerotic plaque burden in mice with a macrophage-specific ablation of ATG5. Together, these data suggested that sequestration of cytotoxic ubiquitinated proteins by p62 protects against atherogenesis, a condition in which the clearance of protein aggregates is disrupted.

Degradation and beyond: the macrophage lysosome as a nexus for nutrient sensing and processing in atherosclerosis.

PURPOSE OF REVIEW: The ability of macrophage lysosomes to degrade both exogenous and internally derived cargo is paramount to handling the overabundance of lipid and cytotoxic material present in the atherosclerotic plaque. We will discuss recent insights in both classical and novel functions of the lysosomal apparatus, as it pertains to the pathophysiology of atherosclerosis. RECENT FINDINGS: Lipid-mediated dysfunction in macrophage lysosomes appears to be a critical event in plaque progression. Consequences include enhanced inflammatory signalling [particularly the inflammasome/interleukin-1ß axis] and an inability to interface with autophagy leading to a proatherogenic accumulation of dysfunctional organelles and protein aggregates. Aside from degradation, several novel functions have recently been ascribed to lysosomes, including involvement in macrophage polarization, generation of lipid signalling intermediates and serving as a nutrient depot for mechanistic target of rapamycin activation, each of which can have profound implications in atherosclerosis. Finally, the discovery of the transcription factor transcription factor EB as a mechanism of inducing lysosomal biogenesis can have therapeutic value by reversing lysosomal dysfunction in macrophages. SUMMARY: Lysosomes are a central organelle in the processing of exogenous and intracellular biomolecules. Together with recent data that implicate the degradation products of lysosomes in modulation of signalling pathways, these organelles truly do lay at a nexus in nutrient sensing and processing. Dissecting the full repertoire of lysosome function and ensuing dysfunction in plaque macrophages is pivotal to our understanding of atherogenesis.

Location-dependent signaling of the group 1 metabotropic glutamate receptor mGlu5.

Although G protein-coupled receptors are primarily known for converting extracellular signals into intracellular responses, some receptors, such as the group 1 metabotropic glutamate receptor, mGlu5, are also localized on intracellular membranes where they can mediate both overlapping and unique signaling effects. Thus, besides "ligand bias," whereby a receptor's signaling modality can shift from G protein dependence to independence, canonical mGlu5 receptor signaling can also be influenced by "location bias" (i.e., the particular membrane and/or cell type from which it signals). Because mGlu5 receptors play important roles in both normal development and in disorders such as Fragile X syndrome, autism, epilepsy, addiction, anxiety, schizophrenia, pain, dyskinesias, and melanoma, a large number of drugs are being developed to allosterically target this receptor. Therefore, it is critical to understand how such drugs might be affecting mGlu5 receptor function on different membranes and in different brain regions. Further elucidation of the site(s) of action of these drugs may determine which signal pathways mediate therapeutic efficacy.

Induction of lysosomal biogenesis in atherosclerotic macrophages can rescue lipid-induced lysosomal dysfunction and downstream sequelae.

OBJECTIVE: Recent reports of a proatherogenic phenotype in mice with macrophage-specific autophagy deficiency have renewed interest in the role of the autophagy-lysosomal system in atherosclerosis. Lysosomes have the unique ability to process both exogenous material, including lipids and autophagy-derived cargo such as dysfunctional proteins/organelles. We aimed to understand the effects of an atherogenic lipid environment on macrophage lysosomes and to evaluate novel ways to modulate this system. APPROACH AND RESULTS: Using a variety of complementary techniques, we show that oxidized low-density lipoproteins and cholesterol crystals, commonly encountered lipid species in atherosclerosis, lead to profound lysosomal dysfunction in cultured macrophages. Disruptions in lysosomal pH, proteolytic capacity, membrane integrity, and morphology are readily seen. Using flow cytometry, we find that macrophages isolated from atherosclerotic plaques also display features of lysosome dysfunction. We then investigated whether enhancing lysosomal function can be beneficial. Transcription factor EB (TFEB) is the only known transcription factor that is a master regulator of lysosomal biogenesis although its role in macrophages has not been studied. Lysosomal stress induced by chloroquine or atherogenic lipids leads to TFEB nuclear translocation and activation of lysosomal and autophagy genes. TFEB overexpression in macrophages further augments this prodegradative response and rescues several deleterious effects seen with atherogenic lipid loading as evidenced by blunted lysosomal dysfunction, reduced secretion of the proinflammatory cytokine interleukin-1ß, enhanced cholesterol efflux, and decreased polyubiquitinated protein aggregation. CONCLUSIONS: Taken together, these data demonstrate that lysosomal function is markedly impaired in atherosclerosis and suggest that induction of a lysosomal biogenesis program in macrophages has antiatherogenic effects.

Self-eating in the plaque: what macrophage autophagy reveals about atherosclerosis.

Autophagy (or 'self-eating') is the process by which cellular contents are recycled to support downstream metabolism. An explosion in research in the past decade has implicated its role in both health and disease and established the importance of the autophagic response during periods of stress and nutrient deprivation. Atherosclerosis is a state where chronic exposure to cellular stressors promotes disease progression, and alterations in autophagy are predicted to be consequential. Recent reports linking macrophage autophagy to lipid metabolism, blunted inflammatory signaling, and an overall suppression of proatherogenic processes support this notion. We review these data and provide a framework for understanding the role of macrophage autophagy in the pathogenesis of atherosclerosis, one of the most formidable diseases of our time.