Metabolic reprogramming in memory CD4 T cell responses of old adults.

To determine whether aging affects the ability of T cells to undergo metabolic reprogramming upon activation, we compared CD4 T cell responses after polyclonal in vitro stimulation. Compared to younger adults, CD4 memory T cells from healthy older individuals exhibited a higher upregulation of oxidative phosphorylation with increased production of reactive oxygen species and intracellular and secreted ATP. Increased ATP secretion led to increased purinergic signaling and P2X7-dependent increases in cytoplasmic calcium. The increased mitochondrial activity was not due to a difference in activation-induced mitochondrial biogenesis. Expression of carnitine palmitoyl transferase 1 was higher, conversely that of fatty acid synthase was reduced in older T cells, resulting in increased fatty acid oxidation, while depleting intracellular lipid stores. The aged CD4 memory T cells therefore maintain a more catabolic state in lipid metabolism, while their ability to upregulate glycolysis upon activation is preserved.

Hypermetabolic macrophages in rheumatoid arthritis and coronary artery disease due to glycogen synthase kinase 3b inactivation.

OBJECTIVES: Accelerated atherosclerotic disease typically complicates rheumatoid arthritis (RA), leading to premature cardiovascular death. Inflammatory macrophages are key effector cells in both rheumatoid synovitis and the plaques of coronary artery disease (CAD). Whether both diseases share macrophage-dependent pathogenic mechanisms is unknown. METHODS: Patients with RA or CAD (at least one myocardial infarction) and healthy age-matched controls were recruited into the study. Peripheral blood CD14+ monocytes were differentiated into macrophages. Metabolic profiles were assessed by Seahorse Analyzer, intracellular ATP concentrations were quantified and mitochondrial protein localisation was determined by confocal image analysis. RESULTS: In macrophages from patients with RA or CAD, mitochondria consumed more oxygen, generated more ATP and built tight interorganelle connections with the endoplasmic reticulum, forming mitochondria-associated membranes (MAM). Calcium transfer through MAM sites sustained mitochondrial hyperactivity and was dependent on inactivation of glycogen synthase kinase 3b (GSK3b), a serine/threonine kinase functioning as a metabolic switch. In patient-derived macrophages, inactivated pGSK3b-Ser9 co-precipitated with the mitochondrial fraction. Immunostaining of atherosclerotic plaques and synovial lesions confirmed that most macrophages had inactivated GSK3b. MAM formation and GSK3b inactivation sustained production of the collagenase cathepsin K, a macrophage effector function closely correlated with clinical disease activity in RA and CAD. CONCLUSIONS: Re-organisation of the macrophage metabolism in patients with RA and CAD drives unopposed oxygen consumption and ultimately, excessive production of tissue-destructive enzymes. The underlying molecular defect relates to the deactivation of GSK3b, which controls mitochondrial fuel influx and as such represents a potential therapeutic target for anti-inflammatory therapy.

Involvement of lysosomal exocytosis in the excretion of mesoporous silica nanoparticles and enhancement of the drug delivery effect by exocytosis inhibition.

The exocytosis of phosphonate modified mesoporous silica nanoparticles (P-MSNs) is demonstrated and lysosomal exocytosis is identified as the mechanism responsible for this event. Regulation of P-MSN exocytosis can be achieved by inhibiting or accelerating lysosomal exocytosis. Slowing down P-MSN exocytosis enhances the drug delivery effect of CPT-loaded P-MSNs by improving cell killing.

Synthesis of biomolecule-modified mesoporous silica nanoparticles for targeted hydrophobic drug delivery to cancer cells.

Synthetic methodologies integrating hydrophobic drug delivery and biomolecular targeting with mesoporous silica nanoparticles are described. Transferrin and cyclic-RGD peptides are covalently attached to the nanoparticles utilizing different techniques and provide selectivity between primary and metastatic cancer cells. The increase in cellular uptake of the targeted particles is examined using fluorescence microscopy and flow cytometry. Transferrin-modified silica nanoparticles display enhancement in particle uptake by Panc-1 cancer cells over that of normal HFF cells. The endocytotic pathway for these particles is further investigated through plasmid transfection of the transferrin receptor into the normal HFF cell line, which results in an increase in particle endocytosis as compared to unmodified HFF cells. By designing and attaching a synthetic cyclic-RGD, selectivity between primary cancer cells (BT-549) and metastatic cancer cells (MDA-MB 435) is achieved with enhanced particle uptake by the metastatic cancer cell line. Incorporation of the hydrophobic drug Camptothecin into these two types of biomolecular-targeted nanoparticles causes an increase in mortality of the targeted cancer cells compared to that caused by both the free drug and nontargeted particles. These results demonstrate successful biomolecular-targeted hydrophobic drug delivery carriers that selectively target specific cancer cells and result in enhanced drug delivery and cell mortality.

AMP-independent activator of AMPK for treatment of mitochondrial disorders.

Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.

Lymphocyte generation and population homeostasis throughout life.

Immune aging is a multi-faceted process that manifests as reduced competence to fight infections and malignant cells, as well as diminished tissue repair, unprovoked inflammation, and increased autoreactivity. The aging adaptive immune system, with its high complexity in functional cell subpopulations and diversity of B- and T-cell receptors, has to cope with the challenge of maintaining homeostasis while responding to exogenous stimuli and compensating for reduced generative capacity. With thymic involution, naïve T cells begin to function as quasi-stem cells and maintain the compartment through peripheral homeostatic proliferation that shapes the T-cell repertoire through peripheral selection and the activation of differentiation pathways. Similarly, reduced generation of early B-cell progenitors alters the composition of the peripheral B-cell compartment with the emergence of a unique, auto-inflammatory B-cell subset, termed age-associated B cells (ABCs). These changes in T- and B-cell composition and function are core manifestations of immune aging.

Epigenomics of human CD8 T cell differentiation and aging.

The efficacy of the adaptive immune response declines dramatically with age, but the cell-intrinsic mechanisms driving immune aging in humans remain poorly understood. Immune aging is characterized by a loss of self-renewing naïve cells and the accumulation of differentiated but dysfunctional cells within the CD8 T cell compartment. Using ATAC-seq, we inferred the transcription factor binding activities correlated with naive and central and effector memory CD8 T cell states in young adults. Integrating our results with RNA-seq, we identified transcription networks associated with CD8 T cell differentiation, with prominent roles implicated for BATF, ETS1, Eomes, and Sp1. Extending our analysis to aged humans, we found that the differences between the memory and naive subsets were largely preserved across age, but that naive and central memory cells from older individuals exhibited a shift toward more differentiated patterns of chromatin openness. Additionally, aged naive cells displayed a loss in chromatin accessibility at gene promoters, largely associated with a decrease in NRF1 binding. This shift was implicated in a marked drop-off in the ability of the aged naive cells to transcribe respiratory chain genes, which may explain the reduced capacity of oxidative phosphorylation in older naïve cells. Our findings identify BATF- and NRF1-driven gene regulation as potential targets for delaying CD8 T cell aging and restoring function.

The glycolytic enzyme PKM2 bridges metabolic and inflammatory dysfunction in coronary artery disease.

Abnormal glucose metabolism and enhanced oxidative stress accelerate cardiovascular disease, a chronic inflammatory condition causing high morbidity and mortality. Here, we report that in monocytes and macrophages of patients with atherosclerotic coronary artery disease (CAD), overutilization of glucose promotes excessive and prolonged production of the cytokines IL-6 and IL-1ß, driving systemic and tissue inflammation. In patient-derived monocytes and macrophages, increased glucose uptake and glycolytic flux fuel the generation of mitochondrial reactive oxygen species, which in turn promote dimerization of the glycolytic enzyme pyruvate kinase M2 (PKM2) and enable its nuclear translocation. Nuclear PKM2 functions as a protein kinase that phosphorylates the transcription factor STAT3, thus boosting IL-6 and IL-1ß production. Reducing glycolysis, scavenging superoxide and enforcing PKM2 tetramerization correct the proinflammatory phenotype of CAD macrophages. In essence, PKM2 serves a previously unidentified role as a molecular integrator of metabolic dysfunction, oxidative stress and tissue inflammation and represents a novel therapeutic target in cardiovascular disease.

Development of mesoporous silica nanomaterials as a vehicle for anticancer drug delivery.

The development of delivery vehicles that would carry therapeutic agents selectively to cancer cells has become an important focus in biomedical research. Nanoparticles have received much attention because the advances made in this field have resulted in multiple biocompatible materials. In particular, mesoporous silica nanoparticles (MSNs) offer a solid framework with porous structure and high surface area that allows for the attachment of different functional groups. In this article we discuss the different surface modifications made to MSNs that have allowed for the construction of targeted nanoparticles to enhance accumulation and uptake in target sites, the incorporation of nanomachines for controlled cargo release and the combination with superparamagnetic metals for MRI cell labeling. We also discuss biocompatibility, biodistribution and drug-delivery efficacy of MSNs. Finally, we mention the construction of multifunctional nanoparticles that combine all of the previously examined nanoparticle modifications.

OKL38 is an oxidative stress response gene stimulated by oxidized phospholipids.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) is present in oxidative modified LDL and accumulates in lesions of many chronic inflammatory diseases, such as atherosclerosis. In a microarray study, OxPAPC has been demonstrated to modulate the expression of >700 genes in human aortic endothelial cells. We found that the levels of mRNA for OKL38 [also named Bone marrow Derived Growth Factor (BDGI)], a tumor growth inhibitor, were strongly increased by OxPAPC. Here, we report that OKL38 is regulated by an oxidative signal induced by OxPAPC and its component lipid 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine. The stimulation of OKL38 by OxPAPC depends on superoxide production, because the NADPH oxidase (Nox) inhibitor apocynin and the superoxide scavenger N-acetyl cysteine block this stimulation. Oxidative stress by tert-butylhydroquinone treatment also induced the expression of OKL38. The stimulation of OKL38 expression by OxPAPC is mediated via transcription factor nuclear factor E2-related factor (Nrf2), a common factor involved in the regulation of oxidative stress-stimulated genes. Activation of Nrf2 induces the expression of OKL38, whereas small interfering RNA knockdown of Nrf2 blocks the stimulation of OKL38 by OxPAPC. Our results suggest that OKL38 is regulated via the Nox/Nrf2 pathway in response to oxidative stress stimuli.