The Effect of Nicotinamide Riboside on LDL-Cholesterol Induced T-Cell Dysfunction
University of Delaware
Aging is associated with chronic low-grade inflammation (“inflammaging”) and is a significant risk factor for multiple chronic diseases, including cardiovascular disease, Alzheimer’s disease, and cancer. Inflammaging is caused by the deterioration of the innate and adaptive immune systems, often accompanied by immunosenescence. T-lymphocytes (T-cells), primarily known for their role in the adaptive immune system responding to foreign antigens, are increasingly recognized as contributors to inflammaging via immunosenescence and impaired mitochondrial function. Age-related declines of cellular nicotinamide adenine dinucleotide (NAD+) levels may be the trigger of immunosenescence and mitochondrial dysfunction. However, the mechanisms leading to T-cell mediated inflammaging and mitochondrial dysfunction are not fully known. One mechanism which might contribute to age related T-cell dysfunction is an increase in endogenous low-density lipoprotein cholesterol (LDL-C) in blood plasma. Increased endogenous LDL-C occurs with aging and has been linked, cross-sectionally, to mitochondrial damage and dysfunction, reduction of ATP synthesis, and increased reactive oxygen species. Subsequently, this damage can lead to various cellular consequences, rapid aging, and disease onset. Supplementation with nicotinamide riboside (NR), a precursor to nicotinamide adenine dinucleotide (NAD+), might be a novel therapeutic to protect T-cells from the deleterious effects of high LDL-C. In addition to its role as a regulator of cellular reoxidation-reduction reactions, NAD+ is a critical co-substrate for several energy-sensing and stress-resistance enzymes. These enzymes are referred to as “NAD+- consuming enzymes” and include the silent mating type information regulation of two homologs (sirtuins; SIRTs), poly adenosine diphosphate (ADP) ribose polymerases (PARPs), cyclic ADP (cADP)-ribose synthases, and CD38/156 ectoenzymes. These enzymes contribute to a multitude of homeostatic processes including the maintenance of biological stress resistance, DNA damage repair, and the regulation of immune cell function. Supplementation with NR has been shown to increase NAD+ bioavailability. NAD+ has emerged as a vital and intriguing cofactor for maintaining mitochondrial fitness by up-regulating enzymes that repair mitochondrial DNA. However, whether NAD+ can protect T-cell mitochondria from immunosenescence is unknown. This study aimed to investigate the efficacy of exogenous NAD+ supplementation in protecting young T-cells from the effects of hypercholesterolemic conditions associated with aging. I hypothesized that treatment with LDL-C would impair T-cell mitochondrial respiration and induce T-cell inflammation and that NR would exert a protective effect on T-cells, preserving mitochondrial respiration and reducing the inflammatory response. Seven adults (6 female/ 1 male) between the ages of 22 and 26 participated in this study. Blood samples were collected in EDTA coated vacutainer tubes from all 7 participants. Peripheral Blood Mononuclear Cells (PBMCs), isolated from whole blood, were further isolated into a pan T-cell sample. The isolated T-cells were later treated and used for the Seahorse XF Analyzer. The supernatant was isolated and frozen to use for the quantification of cytokines. Pan T-cells were treated with high (4.9 mMol/L) physiologic concentrations of LDL-C and co-incubated with high LDL C and NR compared to control (Serum-free media, 0 mMol/L). T-cell mitochondrial function and inflammatory cytokine production were assessed by measuring mitochondrial respiration using an extracellular flux analyzer (Seahorse XFe96 Analyzer) and multi-plex protein quantification (Luminex Magpix), respectively. Concentrations of pro-and anti-inflammatory cytokines, specifically interleukin-6 (IL 6) and interleukin-10 (IL-10), were measured across the three treatment groups of the pan T-cell samples. Proinflammatory cytokines up-regulate the production of radical oxygen species (ROS) and anti-inflammatory cytokines reduce the production of ROS. Contrary to our hypothesis, LDL-C non-significantly augmented T-cell mitochondrial respiration during maximal oxygen consumption rate (OCR), while it non-significantly decreased basal OCR and ATP-linked OCR. Further, the addition of NR to LDL-C treated samples exacerbated mitochondrial respiration in all accounts. Anti-inflammatory cytokine production showed a non-significant decrease with the LDL-C and LDL-C+NR treatments. Proinflammatory cytokine production displayed a non-significant increase within the LDL-C and LDL-C+NR treatment groups. The additional NR treatment to LDL-C treated T-cells significantly decreased the basal, maximum, and ATP-linked OCR, but it is unclear if LDL-C treatment alone had a significant effect on T-cell mitochondrial dysfunction. Cytokine production was not significantly affected with either treatment at the current sample size and may require more samples to clarify a relationship.
Aging , Nicotinamide riboside , T-cells , Lipoprotein cholesterol