Resting natural killer cell homeostasis relies on tryptophan/NAD+ metabolism and HIF-1α.

Natural killer (NK) cells are forced to cope with different oxygen environments even under resting conditions. The adaptation to low oxygen is regulated by oxygen-sensitive transcription factors, the hypoxia-inducible factors (HIFs). The function of HIFs for NK cell activation and metabolic rewiring remains controversial. Activated NK cells are predominantly glycolytic, but the metabolic programs that ensure the maintenance of resting NK cells are enigmatic. By combining in situ metabolomic and transcriptomic analyses in resting murine NK cells, our study defines HIF-1α as a regulator of tryptophan metabolism and cellular nicotinamide adenine dinucleotide (NAD+ ) levels. The HIF-1α/NAD+ axis prevents ROS production during oxidative phosphorylation (OxPhos) and thereby blocks DNA damage and NK cell apoptosis under steady-state conditions. In contrast, in activated NK cells under hypoxia, HIF-1α is required for glycolysis, and forced HIF-1α expression boosts glycolysis and NK cell performance in vitro and in vivo. Our data highlight two distinct pathways by which HIF-1α interferes with NK cell metabolism. While HIF-1α-driven glycolysis is essential for NK cell activation, resting NK cell homeostasis relies on HIF-1α-dependent tryptophan/NAD+ metabolism.

Vaccination-based immunotherapy to target profibrotic cells in liver and lung.

Fibrosis is the final path of nearly every form of chronic disease, regardless of the pathogenesis. Upon chronic injury, activated, fibrogenic fibroblasts deposit excess extracellular matrix, and severe tissue fibrosis can occur in virtually any organ. However, antifibrotic therapies that target fibrogenic cells, while sparing homeostatic fibroblasts in healthy tissues, are limited. We tested whether specific immunization against endogenous proteins, strongly expressed in fibrogenic cells but highly restricted in quiescent fibroblasts, can elicit an antigen-specific cytotoxic T cell response to ameliorate organ fibrosis. In silico epitope prediction revealed that activation of the genes Adam12 and Gli1 in profibrotic cells and the resulting "self-peptides" can be exploited for T cell vaccines to ablate fibrogenic cells. We demonstrate the efficacy of a vaccination approach to mount CD8+ T cell responses that reduce fibroblasts and fibrosis in the liver and lungs in mice. These results provide proof of principle for vaccination-based immunotherapies to treat fibrosis.

The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut.

Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22-producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ-producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ-expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22-inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.

NK cells in hypoxic skin mediate a trade-off between wound healing and antibacterial defence.

During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin.

Nitric oxide restores peripheral blood mononuclear cell adhesion against hypoxia via NO-cGMP signalling.

Hypoxia is the most detrimental threat to humans residing at high altitudes, affecting multifaceted cellular responses that are crucial for normal homeostasis. Inhalation of nitric oxide has been successfully implemented to combat the hypoxia effect in the high altitude patients. We hypothesize that nitric oxide (NO) restores the peripheral blood mononuclear cell-matrix deadhesion during hypoxia. In the present study, we investigate the cellular action of exogenous NO in the hypoxia-mediated diminution of cell-matrix adhesion of PBMNC and NO bioavailability in vitro. The result showed that NO level and cell-matrix adhesion of PBMNC were significantly reduced in hypoxia as compared with normoxia, as assessed by the DAF-FM and cell adhesion assay, respectively. In contrast, cellular oxidative damage response was indeed upregulated in hypoxic PBMNC. Further, gene expression analysis revealed that mRNA transcripts of cell adhesion molecules (Integrin α5 and ß1) and eNOS expressions were significantly downregulated. The mechanistic study revealed that administration of NO and 8-Br-cGMP and overexpression of eNOS-GFP restored the basal NO level and recovers cell-matrix adhesion in PBMNC via cGMP-dependent protein kinase I (PKG I) signalling. In conclusion, NO-cGMP/PKG signalling may constitute a novel target to recover high altitude-afflicted cellular deadhesion. SIGNIFICANCE OF THIS STUDY: Cellular adhesion is a complex multistep process. The ability of cells to adhere to extracellular matrix is an essential physiological process for normal homeostasis and function. Hypoxia exposure in the PBMNC culture has been proposed to induce oxidative damage and cellular deadhesion and is generally believed to be the key factor in the reduction of NO bioavailability. In the present study, we demonstrated that NO donor or overexpression of eNOS-GFP has a protective effect against hypoxia-induced cellular deadhesion and greatly improves the redox balance by inhibiting the oxidative stress. Furthermore, this protective effect of NO is mediated by the NO-cGMP/PKG signal pathway, which may provide a potential strategy against hypoxia.

Protein Phosphatase 1α enhances renal aldosterone signaling via mineralocorticoid receptor stabilization.

Stimulation of the mineralocorticoid receptor (MR) by aldosterone controls several physiological parameters including blood pressure, inflammation or metabolism. We previously showed that MR turnover constitutes a crucial regulatory step in the responses of renal epithelial cells to aldosterone. Here, we identified Protein Phosphatase 1 alpha (PP1α), as a novel cytoplasmic binding partner of MR that promotes the receptor activity. The RT-PCR expression mapping of PP1α reveals a high expression in the kidney, particularly in the distal part of the nephron. At the molecular level, we demonstrate that PP1α inhibits the ubiquitin ligase Mdm2 by dephosphorylation, preventing its interaction with MR. This results in the accumulation of the receptor due to reduction of its proteasomal degradation and consequently a greater aldosterone-induced Na+ uptake by renal cells. Thus, our findings describe an original mechanism involving a phosphatase in the regulation of aldosterone signaling and provide new and important insights into the molecular mechanism underlying the MR turnover.

sFRP4 signalling of apoptosis and angiostasis uses nitric oxide-cGMP-permeability axis of endothelium.

Nitric oxide (NO) plays a critical role in endothelial functions such as cellular migration, vascular permeability and angiogenesis. Angiogenesis, the formation of new blood vessels from "pre-existing" ones is a carefully regulated process and essential during reproduction, development and wound healing. Previously our lab group reported that Secreted Frizzled-Related Protein 4 (sFRP4) could inhibit angiogenesis in both in vitro and in vivo conditions. sFRP4 belongs to a family of secreted glycoproteins that function as antagonists of the canonical Wnt signalling pathway. Although the pro-apoptotic role of sFRP4 is well discussed in literature, little is known in regards to its anti-angiogenic property. The objective of this study was to elucidate sFRP4 implications in NO biology of the endothelium. Results demonstrate that sFRP4 causes endothelial dysfunction by suppressing NO-cGMP signaling and elevating corresponding ROS levels. The imbalance between NO and ROS levels results in apoptosis and subsequent leakiness of endothelium as confirmed in vivo (Texas red/Annxin - CAM assay) and in vitro (Monolayer permeability assay) conditions. Furthermore utilizing peptides synthesized from the CRD domain of sFRP4, our results showed that while these peptides were able to cause endothelial dysfunctions, they did not cause apoptosis of the endothelial cells. Thereby confirming that sFRP4 can mediate its anti-angiogenic effect independent of its pro-apoptotic property. In conclusion, the current study reports that sFRP4-mediated anti-angiogenesis occurs as a result of impaired NO-cGMP signaling which in turn allow for elevation of redox levels and promotion of apoptosis of endothelial cells.

PHD2 Is a Regulator for Glycolytic Reprogramming in Macrophages.

The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1α (HIF-1α) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior.

Cadmium-induced embryopathy: nitric oxide rescues teratogenic effects of cadmium.

Although Cadmium (Cd) is a well-known heavy metal pollutant and teratogen, the mechanism behind Cd-mediated teratogenicity remains unknown. Previously, we have reported of the protective role of Nitric oxide (NO), a key signaling molecule in the embryonic developmental process, against Thalidomide-induced teratogenicity. The objective of this study was to obtain a mechanistic in-sight of the antiteratogenic potential of NO against Cd-mediated teratogenicity. To achieve this goal, we first studied the effect of Cd on the vasculature of developing embryos and then we investigated whether Cd mediated its effects by interfering with the redox regulation of NO signaling in the early development milieu. We used a chick embryonic model to determine the time and dose-dependent effects of Cd and NO recovery against Cd assault. The effects of Cd and NO recovery were assessed using various angiogenic assays. Redox and NO levels were also measured. Results demonstrated that exposure to Cd at early stage of development caused multiple birth defects in the chick embryos. Exposure to Cd suppressed endogenous NO levels and cGMP signaling, inhibiting angioblast activation and subsequently impairing yolk sac vascular development. Furthermore, Cd-induced superoxide and lipid peroxidation mediated activation of proapoptotic markers p21 and p53 in the developing embryo. Cd also caused the down-regulation of FOXO1, and up-regulation of FOXO3a and Caspase 3-mediated apoptosis. Addition of exogenous NO through a NO donor was able to blunt Cd-mediated effects and restore normal vascular and embryonic development. In conclusion, Cd-mediated teratogenicity occurs as a result of impaired NO-cGMP signaling, increased oxidative stress, and the activation of apoptotic pathways. Subsequent addition of exogenous NO through NO donor negated Cd-mediated effects and protected the developing embryo.

Study on paraoxonase 1 in type 2 diabetes mellitus.

Type 2 Diabetes Mellitus (T 2 D M) occurring as a result of reduced insulin action, is seen in a larger section of population. It is also a condition of Oxidative Stress utilizing the antioxidant resources of the body. One such antioxidant is the Paraoxonasel (PON1) enzyme associated with High Density Lipoprotein. So, the activity of PON1 may be reduced in T 2 D M. Hence, this study was taken up to analyze the status of PON I activity in patients with Type 2 Diabetes Mellitus, attending the Diabetic OP of Sree Balaji Medical College and Hospital (SBMCH). The study included 93 Type 2 Diabetic patients and 89 age and sex matched healthy controls. Paraoxonase 1 activity was assayed by Fluorimetry using the Invitrogen Molecular probes kit. There was a significant reduction in PON1 activity (p value > 0.001) along with a decrease in HDL cholesterol among the Type 2 D M patients compared to healthy controls. The progression of Diabetes Mellitus through the years reflected in a much more reduction in PON1 activity as shown by the Pearsons' correlation analysis. The results were analyzed using SPSS statistical package. It is concluded that Type 2 D M being a condition of Oxidative stress has resulted in the reduction of the antioxidant activity of enzyme PON1.

A comparative study of NONOate based NO donors: spermine NONOate is the best suited NO donor for angiogenesis.

Nitric oxide (NO) is a known modulator of angiogenesis. The NONOate subfamily of NO donors has long been used in experimental and clinical studies to promote angiogenesis. However, no studies have been conducted yet to compare the angiogenesis potential of these NO donors in respect to their pattern of NO release. We hypothesize that having different pattern of NO release, each of the NO donors in NONOate subfamily can promote key stages of angiogenesis in differential manner. To verify our hypothesis, NO donors with half life ranging from seconds to several hours and having very different pattern of NO release were selected to evaluate their efficacy in modulating angiogenesis. Endothelial tube formation using EAhy926 cells was maximally increased by Spermine NONOate (SP) treatment. SP treatment maximally induced both ex vivo and in vivo angiogenesis using egg yolk and cotton plug angiogenesis models respectively. Experiment using chick embryo partial ischemia model revealed SP as the best suited NO donor to recover ischemia driven hampered angiogenesis. The present study elaborated that differential release pattern of NO by different NO donors can modulate angiogenesis differentially and also suggested that SP have a unique pattern of NO release that best fits for angiogenesis.

Nitric oxide protects endothelium from cadmium mediated leakiness.

Cadmium targets the vascular endothelium causing endothelial dysfunction and leakiness of endothelial barrier. Nitric oxide plays a major role in mediating endothelial functions including angiogenesis, migration and permeability. The present study investigates the nitric oxide effects on cadmium induced endothelial leakiness. Results of ex vivo and in vitro permeability assays showed that even a sub-lethal dose of cadmium chloride (1 µM) was sufficient to induce leakiness of endothelial cells. Cadmium drastically altered the actin polymerisation pattern and membrane tension of these cells compared to controls. Addition of nitric oxide donor Spermine NONOate (SP) significantly blunted cadmium-mediated effects and recover endothelial cells integrity. Cadmium-induced cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability and high reactive oxygen species generation. In brief, we show the protective role of nitric oxide against cadmium-mediated endothelial leakiness.

Synthesis and anti-angiogenic activity of benzothiazole, benzimidazole containing phthalimide derivatives.

Benzothiazole and benzimidazole containing phthalimide derivatives (NK037, NK041, NK042, NK0139A and NK0148) have been synthesized and their anti-angiogenic activity was evaluated using ex vivo egg yolk angiogenesis model. A comparative study with pure thalidomide (NKTA) has also been performed to describe the efficacy of these derivatives in blocking angiogenesis. NK037, NK041 and NK042 were equally potent in blocking egg yolk angiogenesis and the anti-angiogenesis effect was higher than NKTA suggesting the efficacy of these three derivatives in blocking angiogenesis when compare to control. Other two derivatives NK0139A and NK0148 showed effect less than NKTA and stronger than control in ex vivo angiogenesis.

Nitrites derived from Foneiculum vulgare (fennel) seeds promotes vascular functions.

Recent evidence has demonstrated that nitrites play an important role in the cardiovascular system. Fennel (Foneiculum vulgare) seeds are often used as mouth fresheners after a meal in both the Indian sub-continent and around the world. The present study aims to quantify the nitrite and nitrates in fennel seeds as well as elucidating the effect of fennel derived-nitrites on vascular functions. Results from our study show that fennel seeds contain significantly higher amount of nitrites when compared to other commonly used post-meal seeds. Furthermore our study confirmed the functional effects of fennel derived-nitrites using in vitro and ex vivo models that describe the promotion of angiogenesis, cell migration, and vasorelaxation. We also showed that chewing fennel seeds enhanced nitrite content of saliva. Thus our study indicates the potential role of fennel derived-nitrites on the vascular system.

Nitric oxide rescues thalidomide mediated teratogenicity.

Thalidomide, a sedative drug given to pregnant women, unfortunately caused limb deformities in thousands of babies. Recently the drug was revived because of its therapeutic potential; however the search is still ongoing for an antidote against thalidomide induced limb deformities. In the current study we found that nitric oxide (NO) rescues thalidomide affected chick (Gallus gallus) and zebrafish (Danio rerio) embryos. This study confirms that NO reduced the number of thalidomide mediated limb deformities by 94% and 80% in chick and zebrafish embryos respectively. NO prevents limb deformities by promoting angiogenesis, reducing oxidative stress and inactivating caspase-3 dependent apoptosis. We conclude that NO secures angiogenesis in the thalidomide treated embryos to protect them from deformities.