Immunotherapy for Infarcts: In Vivo Postinfarction Macrophage Modulation Using Intramyocardial Microparticle Delivery of Map4k4 Small Interfering RNA

The myeloid cells infiltrating the heart early after acute myocardial infarction elaborate a secretome that largely orchestrates subsequent ventricular wall repair. Regulating this innate immune response could be a means to improve infarct healing. To pilot this concept, we utilized (beta1,3-d-) glucan-encapsulated small interfering RNA (siRNA)-containing particles (GeRPs), targeting mononuclear phagocytes, delivered to mice as a one-time intramyocardial injection immediately after acute infarction. Findings demonstrated that cardiac macrophages phagocytosed GeRPs in vivo and had little systemic dissemination, thus providing a means to deliver local therapeutics. Acute infarcts were then injected in vivo with phosphate-buffered saline (PBS; vehicle) or GeRPs loaded with siRNA to Map4k4, and excised hearts were examined at 3 and 7 days by quantitative polymerase chain reaction, flow cytometry, and histology. Compared with infarcted PBS-treated hearts, hearts with intrainfarct injections of siRNA-loaded GeRPs exhibited 69-89% reductions in transcripts for Map4k4 (mitogen-activated protein kinase kinase kinase kinase 4), interleukin (IL)-1beta, and tumor necrosis factor alpha at 3 days. Expression of other factors relevant to matrix remodeling-monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases, hyaluronan synthases, matricellular proteins, and profibrotic factors transforming growth factor beta (TGF-beta), and connective tissue growth factor (CTGF)-were also decreased. Most effects peaked at 3 days, but, in some instances (Map4k4, IL-1beta, TGF-beta, CTGF, versican, and periostin), suppression persisted to 7 days. Thus, direct intramyocardial GeRP injection could serve as a novel and clinically translatable platform for in vivo RNA delivery to intracardiac macrophages for local and selective immunomodulation of the infarct microenvironment

Identification of Map4k4 as a novel suppressor of skeletal muscle differentiation

Myoblast differentiation into mature myotubes is a critical step in the development and repair of human skeletal muscle. Here we show that small interfering RNA (siRNA)-based silencing of the Ste20-like mitogen-activated protein 4 kinase 4 (Map4k4) in C2C12 myoblasts markedly enhances expression of myogenic differentiation genes, myoblast fusion, and myotube diameter. In contrast, adenovirus-mediated expression of native Map4k4 in C2C12 cells attenuates each of these processes, indicating that Map4k4 is a negative regulator of myogenic differentiation and hypertrophy. Expression of a Map4k4 kinase-inactive mutant enhances myotube formation, suggesting that the kinase activity of Map4k4 is essential for its inhibition of muscle differentiation. Map4k4 regulation of myogenesis is unlikely to be mediated by classic mitogen-activated protein kinase (MAPK) signaling pathways, because no significant difference in phosphorylation of extracellular signal-regulated kinase (ERK), p38, or c-Jun N-terminal kinase (JNK) is observed in Map4k4-silenced cells. Furthermore, silencing of these other MAPKs does not result in a hypertrophic myotube phenotype like that seen with Map4k4 depletion. Uniquely, Map4k4 silencing upregulates the expression of the myogenic regulatory factor Myf5, whose depletion inhibits myogenesis. Furthermore, Myf5 is required for enhancement of myotube formation in Map4k4-silenced cells, while Myf5 overexpression rescues Map4k4-mediated inhibition of myogenic differentiation. These results demonstrate that Map4k4 is a novel suppressor of skeletal muscle differentiation, acting through a Myf5-dependent mechanism

Accelerated phosphatidylcholine turnover in macrophages promotes adipose tissue inflammation in obesity.

White adipose tissue (WAT) inflammation contributes to the development of insulin resistance in obesity. While the role of adipose tissue macrophage (ATM) pro-inflammatory signalling in the development of insulin resistance has been established, it is less clear how WAT inflammation is initiated. Here, we show that ATMs isolated from obese mice and humans exhibit markers of increased rate of de novo phosphatidylcholine (PC) biosynthesis. Macrophage-specific knockout of phosphocholine cytidylyltransferase A (CCTα), the rate-limiting enzyme of de novo PC biosynthesis pathway, alleviated obesity-induced WAT inflammation and insulin resistance. Mechanistically, CCTα-deficient macrophages showed reduced ER stress and inflammation in response to palmitate. Surprisingly, this was not due to lower exogenous palmitate incorporation into cellular PCs. Instead, CCTα-null macrophages had lower membrane PC turnover, leading to elevated membrane polyunsaturated fatty acid levels that negated the pro-inflammatory effects of palmitate. Our results reveal a causal link between obesity-associated increase in de novo PC synthesis, accelerated PC turnover and pro-inflammatory activation of ATMs

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe(-/-) mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe(-/-) and Ldlr(-/-) mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFkappaB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis

Human resident liver myeloid cells protect against metabolic stress in obesity

Although multiple populations of macrophages have been described in the human liver, their function and turnover in patients with obesity at high risk of developing non-alcoholic fatty liver disease (NAFLD) and cirrhosis are currently unknown. Herein, we identify a specific human population of resident liver myeloid cells that protects against the metabolic impairment associated with obesity. By studying the turnover of liver myeloid cells in individuals undergoing liver transplantation, we find that liver myeloid cell turnover differs between humans and mice. Using single-cell techniques and flow cytometry, we determine that the proportion of the protective resident liver myeloid cells, denoted liver myeloid cells 2 (LM2), decreases during obesity. Functional validation approaches using human 2D and 3D cultures reveal that the presence of LM2 ameliorates the oxidative stress associated with obese conditions. Our study indicates that resident myeloid cells could be a therapeutic target to decrease the oxidative stress associated with NAFLD

Macrophage heterogeneity and energy metabolism

Macrophages are versatile and multifunctional cell types present in most vertebrate tissues. They are the first line of defense against pathogens through phagocytosis of microbial infections, particles and dead cells. Macrophages harbor additional functions besides immune protection by participating in essential homeostatic and tissue development functions. The immune response requires a concomitant and coordinated regulation of the energetic metabolism. In this review, we will discuss how macrophages influence metabolic tissues and in turn how metabolic pathways, particularly glucose and lipid metabolism, affect macrophage phenotypes

The role of MAPKs in adipocyte differentiation and obesity.

The ERK, p38 and JNK mitogen activated protein kinases (MAPKs) are intracellular signalling pathways that play a pivotal role in many essential cellular processes such as proliferation and differentiation. MAPKs are activated by a large variety of stimuli and one of their major functions is to connect cell surface receptors to transcription factors in the nucleus, which consequently triggers long-term cellular responses. This review focuses on their in vitro and in vivo roles in adipocyte differentiation and obesity. Hyperplasia of adipose tissue is a critical event for the development of obesity. Several studies have analysed the role of MAPKs in vitro in adipocyte differentiation of preadipocyte established cell lines. In the case of ERK, although the first data appeared contradictory, a consensus scenario arises: ERK would be necessary to initiate the preadipocyte into the differentiation process and, thereafter, this signal transduction pathway needs to be shut-off to proceed with adipocyte maturation. The limitation of these cellular models is that only terminal adipocyte differentiation can be analysed, eluding the early proliferative steps of adipogenesis. New insights are now emerging by investigations conducted either in vitro with the use of embryonic stem (ES) cells or in vivo with mice where these genes are invalidated. These studies not only confirm and/or precise the various functions of MAPKs in adipogenesis but, importantly, reveal unsuspected roles, for example JNK in obesity or ERK in adipogenesis of ES cells, and, for a given pathway, assign specific functions to each isoform. It appears now that a fine tuning of the MAPKs regulates both normal and pathological adipogenesis. The precise understanding of the cascade of these molecular events and the way to regulate them will be certainly crucial in order to efficiently fight obesity

Insulin signalling mechanisms for triacylglycerol storage

Insulin signalling is uniquely required for storing energy as fat in humans. While de novo synthesis of fatty acids and triacylglycerol occurs mostly in liver, adipose tissue is the primary site for triacylglycerol storage. Insulin signalling mechanisms in adipose tissue that stimulate hydrolysis of circulating triacylglycerol, uptake of the released fatty acids and their conversion to triacylglycerol are poorly understood. New findings include (1) activation of DNA-dependent protein kinase to stimulate upstream stimulatory factor (USF)1/USF2 heterodimers, enhancing the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c); (2) stimulation of fatty acid synthase through AMP kinase modulation; (3) mobilisation of lipid droplet proteins to promote retention of triacylglycerol; and (4) upregulation of a novel carbohydrate response element binding protein beta isoform that potently stimulates transcription of lipogenic enzymes. Additionally, insulin signalling through mammalian target of rapamycin to activate transcription and processing of SREBP1c described in liver may apply to adipose tissue. Paradoxically, insulin resistance in obesity and type 2 diabetes is associated with increased triacylglycerol synthesis in liver, while it is decreased in adipose tissue. This and other mysteries about insulin signalling and insulin resistance in adipose tissue make this topic especially fertile for future research

Regulation of ES Cell Lineage Commitment by Mitogen Activated Protein Kinases: MAPKs and ES cell differentiation

Embryonic Stem (ES) cells can give rise, in vivo, to the ectodermal, endodermal and mesodermal germ layers and, in vitro, can differentiate into multiple cell lineages, offering broad perspectives in regenerative medicine. Understanding the molecular mechanisms governing ES cell commitment is an essential challenge in this field. The Mitogen Activated Protein Kinase (MAPK) pathways, ERK, JNK and p38MAPK, are able to regulate ES commitment from early steps of the process to mature differentiated cells. Whereas the ERK pathway inhibits the self-renewal of ES cells, upon commitment this pathway is involved in the development of extraembryonic tissues, in early mesoderm differentiation and in the formation of mature adipocytes; p38MAPK displays a large spectrum of action from neurons to adipocytes and JNK is involved in both ectoderm and primitive endoderm differentiations. Furthermore, for a given pathway, several of these effects are isoform-dependent, revealing the complexity of the cellular response to activation of MAPK pathways. Regarding tissue regeneration, the potential outcome of systematic analysis of the function of different MAPKs in different ES cell differentiation programs is discussed