Modulation of Host Immunity with Dietary Supplements Nicotinamide

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Abstract

Immune activation and suppression respectively protect the host from disease and inflammatory damage. An imbalance in these responses, as identified in sepsis, can lead to excessive inflammation, tissue injury, disease progression, secondary infections, organ failure, and death. Macrophages are early responders to invading pathogens and are identified in nearly every organ. The mechanisms that regulate the activity of macrophages are therefore important to infection, inflammation and disease progression. The hypoxia inducible transcription factor (HIF)- 1 alpha regulates anaerobic metabolism and is linked to pro-inflammatory responses in myeloid cells. Specifically, HIF-1 alpha promotes neutrophil (PMN) migration, macrophage phagocytosis, and antigen presentation. The Gram-negative outer membrane molecule, lipopolysaccharide (LPS), induces the production and stabilization of HIF-1 alpha through the activation of the HIF-1 alpha transcription factor, NF-kappa B, and by modulating enzymes involved in HIF-1-alpha degradation, respectively. LPS also induces the expression of macrophage antigen presentation molecules (CD40, CD80), checkpoint molecules (programmed death ligand (PD-L1)), and the nicotinamide adenine dinucleotide (NAD) consuming enzyme, CD38. Nicotinamide (NAM) is the amide version of Vitamin B3 and the primary source of NAD. This molecule is used in NAD(P)H redox reactions or consumed as a substrate by poly(ADP-ribose) polymerases (PARPs), CD38 and sirtuins. In vitro and in vivo, NAM is identified in modulating the function of myeloid cell subsets. The effects of NAM on LPS-induced HIF-1 alpha 1 stabilization, activity, and subsequent immunological responses in macrophages have not been significantly explored. We therefore examined the mechanisms of NAM in regulating LPS-activated human monocyte-derived macrophages (HMDMs). The effects of NAM in this study are expected to offer insight into therapies that target LPS-induced molecules (CD38, PD-L1), hypoxic metabolism, and innate responses to infection. Specific Aim 1: Determine whether NAM treatment alters hypoxic cell signals in human monocyte-derived macrophages (HMDMs) by: - Probing for HIF-1 alpha protein levels, hypoxia response element transcriptional activity, and global gene expression changes via microarray analysis. - Confirming possible changes at the gene level with protein assays. - Determining whether exposure to NAM alters HMDM function, by probing phagocytosis and antigen presentation ability. Current Status: A microarray of HMDMs derived from 4 healthy donors and treated in vitro with control, NAM, LPS, and LPS+NAM was performed. Principal component analysis of the results revealed global shifts in transcriptional profiles in response to the 4 treatment conditions. HIF-1 alpha expression was lower in the microarray assessment of the NAM effect in the LPS response and this effect was supported by reporter assays and immunoblots that identified reduced HIF-1 alpha transcription and protein, respectively. LPS-induced expression of CD40, CD80, CD38, and PD-L1 were also antagonized by NAM in the microarray, RT-PCR assays, and by flow cytometry. Macrophage endocytosis of dextran and phagocytosis of pseudomonas aeruginosa were also inhibited by NAM treatment. Specific Aim 2: Determine whether NAM treatment alters myeloid cell metabolism by: - Exploring the redox potential (NAD/NADH) of HMDMs upon exposure to NAM. - Determining the preferred metabolic pathways (lactate production and glucose uptake) of HMDMs upon NAM treatment. - Probing global metabolomic changes. Current Status: To understand the mechanisms of the NAM effect we compared NAM treatment to a known antioxidant, N-acetyl cysteine (NAC). This molecule is a precursor to cysteine in the synthesis of glutathione, which detoxifies the reactive oxygen species H2O2 into H2O. NAC comparably inhibited HIF-1 alpha and reduced the cell surface expression of CD38, PD-L1 and CD40. NAC also inhibited LPS-induced H2O2 production whereas NAM enhanced the production, suggesting that NAM does not function as an antioxidant, like NAC, particularly with respect to the reactive oxygen species H2O2. Because NAM is a primary component of NAD, we assessed if NAM altered the NAD/NADH ratio in HMDMs. We identified an increase in the NAD/NADH ratio in NAM treated HMDMs, primarily through the salvage pathway and not the de novo pathway as indicated by probing via immunoblots for the rate-limiting enzymes in each pathway. We additionally examined the microarray for the expression of NAD consuming genes, specifically PARPs and sirtuins. The NAM effect in the LPS response for these genes revealed increased expression of sirtuins but decreased expression of PARPs. NAD also fuels the citric acid cycle and generates alpha-ketoglutarate that is essential for prolyl hydroxylase domain (PHD)-2 hydroxylation of HIF-1 alpha and subsequent von Hippel-Lindau (VHL)-associated HIF-1 alpha ubiquitination for targeted proteasome degradation. In NAM treated HMDMs, we identified increased PHD2 production, unchanged VHL, and decreased PMSF1, which blocks the assembly of the 19S structure in the 26S proteasome. Moreover, the proteasome inhibitor, MG132, effectively blocked NAM inhibition of HIF-1 alpha indicating that NAM functions, in part, through the proteasome. NAM also induced increases in the production of E3 ligases (SART1, FBXW7, STUB1), which serve as a platform for HIF-1 alpha ubiquitination and targeted degradation in either the lysosome or proteasome. Probing the microarray for ubiquitin genes revealed distinct shifts in response to LPS and NAM. Immunoprecipitation of HIF-1 alpha revealed the ubiquitination of this protein in the presence of NAM, bolstering the idea that NAM-induces the expression of enzymes and post-translational responses involved in the degradation of HIF-1 alpha. Specific Aim 3: Design studies to unravel possible mechanisms of NAM modulation of immune status. Current Status: NAM has also been characterized to inhibit the activity of p65/RelA in macrophages and this NF-Kappa B dimer unit regulates the transcription of HIF-1 alpha. The activity of p65/RelA is regulated by the deacetylases HDAC3 and sirtuin 1 (SIRT1), which were both upregulated in the NAM plus LPS treatment compared to LPS alone in our microarray. We therefore performed immunoprecipitations of p65 from HMDM lysates and probed for acetylated-p65 and identified reduced acetylation in the presence of NAM. Because acetylation is important to the transcriptional activity of p65, these data correlated with the reduced levels of GFP-p65 and mCherry-TNF reporters identified by flow cytometry in murine macrophages treated with NAM. We also measured the metabolic effects of NAM in Seahorse assays, which measure cellular oxygen and proton levels in response to nutrient stress and/or cell stimulation. Preliminary data indicate that NAM treatment inhibits glycolysis as well as non-glycolytic acidification where the latter may include protons from the citric acid cycle or the breakdown of intracellular glycogen. Additional metabolomic profiling of HMDMs identified increased production of plasmalogens and activation of the hexosamine biosynthetic pathway. Overall, this study has relevance in the context of inflammatory disorders where excessive inflammation is thought to play a role in the pathogenesis of disease, such as sepsis, autoimmunity, or immune-related adverse events. This year, additional experiments were conducted to confirm some earlier findings. Manuscript is being prepared for submission. Projected Publication: Nicotinamide regulates LPS-induced HIF-1alpha responses through post-translational modifications in human macrophages. Colleen S. Curran, Edward J. Dougherty, Cumhur Yusuf Demirkale, Parizad Torabi-Parizi