SUMMARY Activation of the transcription factor NF-κB is essential to innate immune function and requires strict regulation. The micronutrient zinc modulates proper host defense and zinc deficiency is associated with elevated inflammation and worse outcomes in response to bacterial infection and sepsis. Previous studies suggest that zinc may regulate NF-κB activity during innate immune activation but a mechanistic basis to support this is lacking. Herein we report that the zinc transporter, SLC39A8 (ZIP8), is a transcriptional target of NF-κB and functions to negatively regulate pro-inflammatory responses through zinc-mediated down-modulation of IKK activity in vitro. Accordingly, fetal fibroblasts obtained from Slc39a8 hypomorphic mice exhibited dysregulated zinc uptake and increased NF-κB activation. Consistent with this, mice provided zinc-deficient dietary intakes developed excessive inflammation to polymicrobial sepsis in conjunction with insufficient control of IKK. Our findings identify a novel negative feedback loop that directly regulates innate immune function through coordination of zinc metabolism.

SummaryBlood transfusion therapy is life-saving for patients with b-thalassaemia and sickle cell disease (SCD), but often results in severe iron overload. This pilot study examined whether the biomarkers of tissue injury or inflammation differ in these two diseases. Plasma malondialdehyde (MDA) was significantly increased 1AE8-fold in thalassaemia relative to control patients. In contrast, MDA in SCD was not significantly different from controls. In multivariate analysis, the strongest predictors of elevated MDA were liver iron concentration (P < 0AE001) and specific diagnosis (P ¼ 0AE019). A significant 2-fold elevation of non-transferrin bound iron (NTBI) was observed in thalassaemia relative to SCD. NTBI was not a significant predictor of high MDA in multivariate analysis. SCD patients showed a significant 2AE2-fold elevation of the inflammatory marker interleukin (IL)-6 relative to controls, and a 3AE6-and 1AE7-fold increase in IL-5 and IL-10 relative to thalassaemia. Although a-tocopherol was significantly decreased by at least 32% in both thalassaemia and SCD, indicating ongoing oxidant stress and antioxidant consumption, c-tocopherol, a nitric oxide-selective antioxidant, was increased 36% in SCD relative to thalassaemia. These results demonstrate that thalassaemia patients have increased MDA and circulating NTBI relative to SCD patients and lower levels of some cytokines and c-tocopherol. This supports the hypothesis that the biology of SCD may show increased inflammation and increased levels of protective antioxidants compared with thalassaemia.

We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl-Lcarnitine (ALCAR) and͞or the mitochondrial antioxidant precursor R-␣-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However, in vitro treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity, thus mimicking agerelated change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction.

Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimer's disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process.iron ͉ complex IV ͉ amyloid precursor protein (APP) ͉ nitric oxide synthase ͉ oxidative stress

Lithium (Li؉ ) has been used to treat mood affect disorders, including bipolar, for decades. This drug is neuroprotective and has several identified molecular targets. However, it has a narrow therapeutic range and the one or more underlying mechanisms of its therapeutic action are not understood. Here we describe a pharmacogenetic study of Li ؉ in the nematode Caenorhabditis elegans. Exposure to Li ؉ at clinically relevant concentrations throughout adulthood increases survival during normal aging (up to 46% median increase). Longevity is extended via a novel mechanism with altered expression of genes encoding nucleosome-associated functions. Li ؉ treatment results in reduced expression of the worm ortholog of LSD-1 (T08D10.2), a histone demethylase; knockdown by RNA interference of T08D10.2 is sufficient to extend longevity (ϳ25% median increase), suggesting Li ؉ regulates survival by modulating histone methylation and chromatin structure.

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier-permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.

This study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted free radical production in primary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Production of reactive species was detected by fluorescence microscopy with the probe 2', 7'-dichlorodihydrofluorescein, which is converted to the fluorescent dichlorofluorescein (DCF) in the presence of various oxidants. Flushing the airspace above the PASMC cultures with normoxic gas (20% O(2), 75% N(2), and 5% CO(2)) resulted in stable PO(2) values of approximately 150 Torr, whereas perfusion of the airspace with hypoxic gas (0% O(2), 95% N(2), and 5% CO(2) ) was associated with a reduction in PO(2) values to stable levels of approximately 25 Torr. Hypoxic PASMCs became increasingly fluorescent at approximately 500% above the normoxic baseline after 60 min. Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea and catalase. These findings show that PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence, suggestive of reactive oxygen or nitrogen species production.

Hypoxia, a stimulus for angiogenesis and vascular remodeling, has been proposed to use reactive oxygen species as second messengers in signal transduction. This contention remains controversial, in part because of vagaries associated with fluorescence‐based methods of free‐radical detection. We took a different approach. Rat main pulmonary artery endothelial cells (PAECs) were cultured in hypoxia for up to 48 h, and, with dichlorofluorescein fluorescence to detect free‐radical production, we used quantitative Southern blot and ligation‐mediated PCR analyses to search for oxidative modifications in the mitochondrial genome and in the nuclear vascular endothelial cell growth factor (VEGF) gene. In accord with previous studies in other cell types, we found that acute hypoxic exposure promoted time‐dependent dichlorofluorescein fluorescence in PAECs. Quantitative Southern blot analysis showed that although hypoxia failed to alter mitochondrial DNA integrity, prominent oxidative lesions occurred in a 5.0‐kb sequence of the VEGF promoter. Using ligation‐mediated PCR to map the modifications at single nucleotide resolution, we found clusters of oxidized bases in a VEGF promoter sequence that included the AP‐1 and HIF‐1 response elements. These actions of hypoxia differed from exogenous xanthine oxidase, which obliterated the mitochondrial genome but failed to erode integrity of the VEGF promoter. Our observations indicate that hypoxia promotes an oxidant stress in main PAECs as detected by oxidative base modifications in the nuclear VEGF gene. The presence of hypoxia‐induced, oxidative base modifications in functionally significant sequences within the VEGF promoter suggests new concepts for mechanisms by which reactive oxygen species participate in hypoxic signal transduction.