Atherosclerosis, the underlying cause of myocardial infarction and stroke, is an inflammatory disease process, and both the innate and adaptive immune responses play important roles in its pathogenesis (1). Monocytes and macrophages play key roles in the pathogenesis of atherosclerosis. Furthermore, alterations in macrophage phenotypes and survival play crucial roles in the development of atherosclerosis. Current studies of immunotherapy have focused on immune checkpoint therapy targeting regulatory pathways in T-cells (2). However, there are also multiple ways to modulate the immune response by changing the activation of myeloid cells, which belong to the innate immune system, performing essential roles in tissue homeostasis by initiating, sustaining, or inhibiting adaptive immunity (3, 4). Therefore, identification of mechanisms that control macrophage metabolism and survival should provide insights into the regulation of immune responses and identify novel approaches to treat disease (5).

Recent studies suggest that interplay between protein kinase B (or Akt) and mammalian target of rapamycin (mTOR) pathways is crucial for myeloid cell activation (4). Akt is a serine/threonine-specific kinase that regulates multiple cellular processes, including cell viability, proliferation, transcription, and migration (6, 7). mTOR is an evolutionarily conserved serine/threonine kinase that is constitutively expressed and plays a central role in integrating environmental cues in the form of growth factors, amino acids, and energy (8). mTOR also controls and shapes the effector responses of innate immune cells. Therefore, knowledge of how Akt/mTOR signaling coordinates the responses of myeloid cells may provide important insights into immunity in health and disease (3, 4).

There are two distinct types of mTOR complexes, termed mTORC1 and mTOR complex 2 (mTORC2), the first contains regulatory associated protein of mTOR (Raptor) and the second involves rapamycin-insensitive companion of mTOR (Rictor) (9). Inhibition of mTORC1 with a class of macrolide immunosuppressive drugs, called rapalogs, reduces atherosclerosis in a variety of animal models, and rapalog eluting stents are widely used to treat atherosclerotic coronary artery disease in humans (10). By contrast, relatively little is known about the role of mTOR2 in atherosclerosis.

Genetic loss of Rictor inactivates mTORC2, and, since total Rictor deficiency in mice causes embryonic lethality (11, 12), generation of mice with tissue-specific knockouts of Rictor has been instrumental in delineating the specific roles of mTORC2 in different cell types. For example, liver-specific disruption of Rictor induces glucose intolerance, hepatic insulin resistance, and decreased hepatic lipogenesis (13, 14). Remarkably, the liver-specific knockout or an inducible total body loss of Rictor in adult mice specifically reduces male lifespan (15). In contrast, Rictor knockout in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity (16). mTORC2 regulates cardiomyocyte growth and survival, and loss of Rictor in mouse heart results in progressive cardiac dysfunction (17). Adipose-specific knockout of Rictor in mice increases whole-body size (18) but leads to hepatic steatosis and insulin resistance (19). Loss of Rictor in pancreatic beta cells reduces beta cell mass and insulin secretion resulting in hyperglycemia and glucose intolerance (20). These data indicate that mTORC2 may play varied roles depending on the cell type and conditions.

Notably, mTORC2 initiates the phosphorylation of Akt S⁴⁷³ (21), which accelerates Akt signaling promoting cell survival and proliferation. Despite the predominant role of mTORC2 as a regulator of pro-survival Akt signaling, the impact of mTORC2 on cell viability remains unclear (22). For example, several studies showed no indication that loss of Rictor and consequently mTORC2 increased apoptosis, at least under otherwise normal physiological conditions (16, 18, 20, 23). Moreover, a recent report indicates that Rictor-deficient keratinocytes display increased lifespan, protection from senescence, and enhanced tolerance of cellular stressors such as growth factor deprivation (24). These findings appear to contradict several other reports indicating that Rictor deficiency increases apoptosis in pulmonary arterial vascular smooth muscle cells (25), endothelial cells (26) and skin tumor cells (27). Rictor plays an important role in regulating cellular survival after ischemic cardiac damage (28) and in B-cells acting via a c-Myc-dependent pathway (29). In addition, we recently demonstrated that loss of IκB kinase alpha (IKKα) in macrophages suppresses Akt S⁴⁷³ phosphorylation and this compromises cell survival and decreases early atherosclerosis (30). This suppression of p-Akt S⁴⁷³ was mediated via mTORC2 suggesting the hypothesis that mTORC2 initiates important pro-survival signaling in monocytes and macrophages that impacts the pathogenesis of atherosclerosis. However, IKKα is located upstream of the NF-κB signal transduction cascade and loss of IKKα-enhanced macrophage activation (31), limiting the ability to draw conclusions regarding the role of mTORC2 in macrophage function and atherogenesis.

Therefore, we used myeloid lineage-specific Rictor deletion in mice to examine directly whether loss of mTORC2 in myeloid cells impacts monocyte and macrophage viability and proliferation and the development of atherosclerosis. We found that mTORC2 signaling is crucial for monocyte and macrophage proliferation and survival, and loss of mTORC2 signaling in myeloid cells results in decreased early atherosclerosis.

The mTOR pathway plays a central role in sensing environmental cues and regulating cell growth, metabolism (8) and immune responses (3, 4). However, the roles of mTORC2 signaling in monocyte/macrophage proliferation and survival, and in atherogenesis remain unclear. Here we generated myeloid lineage-specific Rictor deletion in mice to demonstrate for the first time that mTORC2 signaling is crucial for proliferation and survival of monocytes and macrophages. Rictor deficiency prevents mTORC2 assembly and this causes a dramatic reduction of insulin-mediated Akt S⁴⁷³ phosphorylation in macrophages. In addition, suppression of Akt signaling was observed in monocytes and neutrophils of MRictor^−/− mice in vivo. Ablation of mTORC2 signaling inhibited monocyte and macrophage proliferation. Furthermore, Rictor deficiency compromised monocyte and macrophage viability, and these dramatic changes in monocyte/macrophage survival were associated with dramatic reductions of atherosclerosis in male and female Ldlr^−/− mice in vivo.

Mammalian target of rapamycin complex 2 regulates cellular metabolism and previous studies have shown that cell-specific Rictor ablation significantly suppresses proliferation of endothelial cells (26), helper T-cells (44) and pancreatic β-cells (20). Our results are consistent with these data and indicate that loss of Rictor in monocytes and macrophages suppresses their proliferation causing a diminished response to a proliferative factor, PDGF. Surprisingly, Oh and co-workers (45) recently reported that mTORC2 ablation increased proliferation in peritoneal resident macrophages compared to wild-type cells. This discrepancy with our results may be explained by the fact that we analyzed peritoneal macrophages originating predominantly from monocytes, whereas they focused on tissue-resident peritoneal macrophages. The tissue-resident peritoneal macrophages express F4/80 and GATA6 transcription factor, and they presumably originated from the yolk sac (46). In our experiment, only a small fraction (5%) of peritoneal macrophages expressed F4/80 after two days in culture. Moreover, the same group also showed that Rictor null monocyte-derived peritoneal macrophages have less proliferation than wild-type cells (45) and this finding is consistent with our results.

Although loss of Rictor in macrophages appears to only modestly affect basal Akt activity, a fact that has been noted previously in several different types of cells (16, 18, 20, 23), the response to insulin was significantly suppressed in MRictor^−/− compared to Rictor^fl/fl macrophages (Figures 1A–C). Moreover, there was an increase in apoptotic monocytes and neutrophils in blood of MRictor^−/− mice (Figures 3A,B). More importantly, our in vitro experiments convincingly demonstrated that MRictor^−/− monocytes and macrophages exhibit compromised viability and generate more apoptosis under conditions of ER stress than Rictor^fl/fl cells (Figures 3C–F). These data support the idea that mTORC2 with Akt S⁴⁷³ phosphorylation are essential for pro-survival signaling (6) and that the failure of this response under conditions of ER stress leads to early apoptosis. Previously we have shown that IKKα^−/− macrophages exhibited a similar defect in Akt S⁴⁷³ phosphorylation and a related increase of apoptosis (30). It is known that IKKα interacts with Rictor and that chemical inhibition or genetic suppression of IKK significantly diminishes mTORC2 activity and Akt signaling (47). Low levels of white blood cells and reduced proliferation of macrophages have been associated with reduced atherosclerosis. Interestingly, proliferation of macrophages in the artery wall has been reported to be an important determinant of atherogenesis (48). Our current findings establish that MRictor^−/− mice exhibited relatively low levels of white blood cells, and this is due, at least in part, to decreased proliferation and compromised viability of both monocytes and neutrophils. Furthermore, we cannot exclude the possibility that MRictor^−/− neutrophils or monocytes may have an inefficient or delayed response for mobilization from the bone marrow. It remains unclear whether the reduced numbers of T- and B-cells in the blood of MRictor^−/− mice are causally related to the reduced numbers or functions of myeloid cells.

Macrophages may be polarized into two distinct subtypes including classically activated M1 and alternatively stimulated M2 macrophages (49, 50). A recent concept of the immune response proposes that Akt-mTORC1 signaling controls polarization by changing metabolism of macrophages (5, 51, 52). For example, LPS treatment induces a switch to high aerobic glycolysis, fatty acid synthesis and a truncated citric acid cycle (the Krebs cycle) to form the M1 pro-inflammatory phenotype. In contrast, IL-4 induces oxidative phosphorylation to generate M2 macrophages for tissue remodeling, immunosuppression and phagocytosis. Both of these shifts generate many metabolites that are acting as signaling molecules with consequent alteration of the downstream immune response. In this context, mTORC1 is considered as a major mediator of the cellular response to stress (53). Loss of tuberous sclerosis 1 leads to constitutive mTORC1 activation in macrophages making them resistant to M2 polarization and producing an increased inflammatory response (54). Accordingly, Raptor deficiency in macrophages reduced chemokine gene expression (55) and loss of mTORC1 in hematopoietic cells impaired myelopoiesis at steady state with dampening of some innate immune responses (56). Thus, Akt-mTORC1 signaling is coupled to metabolic input to regulate a key enzyme of Acetyl-CoA synthesis, leading to increased histone acetylation and induction of a subset of M2 genes in macrophages (57).

Compared to mTORC1, our understanding of the role of mTORC2 in macrophage polarization is less well defined. Brown and co-workers (58) were the first to demonstrate that mTORC2 negatively regulates the Toll-like receptor 4-mediated inflammatory response acting via Forkhead box protein O1, a transcription factor regulating glucose metabolism. Festuccia and co-authors later reported that Rictor deletion induces M1 macrophage polarization potentiating pro-inflammatory responses (41). More recent studies revealed that loss of Rictor inhibits the generation of M2 macrophages while preserving the production of classically activated M1 cells (42). Furthermore, mTORC2 operates in parallel with the STAT6 pathway to facilitate increased glycolysis during M2 activation (43).

Depending on the degree of activation, PI3K-Akt signaling crucially contributes to macrophage polarization with subsequent activation or dampening the immune response (59). The individual Akt isoforms promote distinct polarization phenotypes in macrophages. For example, macrophage Akt1 ablation induces M1, whereas Akt2 deficiency promotes the M2 phenotype (60). Recently we have shown that Akt2^−/− macrophages express significantly lower levels of inflammatory genes and display the M2 phenotype with a suppressed ability for M1 polarization and CCR2 induction (61). In addition to the ratio of Akt1/Akt2 isoforms, the levels of Akt signaling are also crucial for macrophage polarization. For example, overexpression of the PI3K-Akt pathway caused by myeloid cell-specific deficiency of SH2-containing inositol phosphatase or phosphatase and tensin homolog (PTEN), which are negative regulators of PI3K signaling, significantly promote M2 macrophage differentiation decreasing inflammatory cytokine production in macrophages (62–64). In contrast, genetic deficiency of IKKα as well as pharmacologic inhibition of IKK, significantly reduces Akt S⁴⁷³ phosphorylation via suppression of mTORC2 signaling, and this is accompanied by an M1-related increase in the expression of inflammatory genes (30). Our current results are consistent with these data and indicate that suppression of Akt signaling increases of inflammatory gene expression in MRictor^−/− macrophages. Moreover, further suppression of signaling with a low dose of Akt inhibitor IV additionally increased cytokine gene expression levels. Importantly, the genetic loss of Raptor, the main component of mTORC1, eliminated this increase, indicating that mTORC1 mediates the increased expression of these pro-inflammatory genes. Together these data support the concept that the PI3K-Akt-mTOR pathway is critical for restriction of the inflammatory reaction and that overexpression of this signaling pathway reduces macrophage activation by LPS, whereas the inhibition of Akt signaling significantly augments NF-κB activity (65). Furthermore our results suggest an opportunity to pharmacologically control Akt signaling activity and polarization of macrophages in order to modulate innate or adaptive immune responses.

Atherosclerosis remains the number one cause of death and disability in modern societies. Surprisingly, relatively little is known about the role of mTORC2 in atherosclerosis. One can propose mechanisms by which loss of mTORC2 might promote atherosclerosis, including increased inflammatory cytokines in response to LPS or increased production of damage-associated molecular patterns; whereas reduced proliferation and increased apoptosis of macrophages would be predicted to reduce the extent of early atherosclerosis. Therefore, we examined the impact of mTORC2 elimination in hematopoietic myeloid cells on atherosclerosis in vivo. MRictor^−/− → Ldlr^−/− recipients had smaller atherosclerotic lesions enriched in apoptotic cells compared to Rictor^fl/fl → Ldlr^−/− mice. Thus, loss of Rictor in hematopoietic myeloid cells significantly suppressed atherosclerosis in Ldlr^−/− mice, and these results demonstrate for the first time that macrophage mTORC2 plays a critical role of in atherogenesis. The reduction of atherosclerotic lesions was likely mediated through several inter-related mechanisms such as the inhibition of proliferation and suppression of viability of monocytes that cause monocytopenia in MRictor^−/− → Ldlr^−/− mice. In addition, we found decreased proliferation and viability of MRictor^−/− macrophages, which are the major type of cells atherosclerotic lesions. These results are consistent with the current concepts that low numbers of circulating monocytes and high sensitivity of macrophages to pro-apoptotic stimuli are key drivers that suppress atherogenesis (66, 67). Our study also suggests a potential role of targeted inhibition of mTORC2 in monocytes and macrophages as a novel approach for the treatment of atherosclerotic cardiovascular disease.

VB. designed and performed experiments, analyzed data, and wrote the manuscript; JH, LD, and YZ performed experiments and analyzed data; JM critically revised the manuscript; and ML supervised the project, designed the studies, analyzed data, and revised the manuscript.

The authors declare that our research was conducted and performed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.