Inflammation alters iron distribution in bone and spleen in mice.

Anemia of inflammation (or inflammation-associated anemia) decreases the quality of life in billions of patients suffering from various inflammatory diseases, such as infection, autoimmune diseases, and cancer, associated with a prolonged state of immune activation. While proper utilization of iron, a nutrient metal essential for erythropoiesis, is important for the prevention of anemia, the alteration of body iron homeostasis upon inflammation, which can contribute to the development of anemia, is not completely understood. Thus, we sought to examine temporal and spatial changes in the distribution of iron and iron-associated molecules during inflammation in mice. To induce inflammation, C57BL/6J mice were injected with turpentine oil weekly for 3 weeks, which resulted in anemia, decreased protein expression of ferroportin, a cellular iron exporter, in the spleen, duodenum, and liver, and increased iron stores in the duodenum and spleen. Tracer kinetic studies after oral administration of 59Fe revealed that more iron was found in the spleen and less in the femur bone in turpentine oil-injected mice compared to the saline-injected mice, indicating tissue-specific abnormalities in iron distribution during inflammation. However, there was no difference in the utilization of iron for red blood cell production after turpentine oil injection; instead, serum hemopexin level and lactate dehydrogenase activity were increased, suggesting increased red blood cell destruction upon inflammation. Our findings provide an improved understanding of temporal and spatial changes in the distribution and utilization of iron during inflammation.

Intracellular Dynamics-Resolved Label-Free Scattering Reveals Real-Time Metabolism of Single Bacteria.

Nanoscopic investigation of bacterial cells is essential to reveal their physiological status, impacting all cellular functions. Currently, this requires labeled probes or targeted staining procedures. Herein, we report a new bacterial feature, intracellular dynamics-resolved Rayleigh scattering (IDRS), that visualizes spatiotemporal cytoplasmic transitions in unlabeled bacteria and characterizes their real-time physiological status in 10 s. From single-bacterium IDRS signals, we discovered unique spatial patterns and their multiple transitions in Gram-negative and Gram-positive bacteria. The magnitude of IDRS signal variation highly correlated with the metabolic status of bacteria, differentiating persistent subpopulations. This is also the first report demonstrating distinct real-time metabolic conditions of unlabeled drug-resistant bacteria that are exposed to different doses of antibiotics. Our strategy opens up a way to simultaneously trace in situ metabolic and antibiotic resistance statuses, which can be applied in single-cell level control of bacterial metabolism and efficacy with a heterogeneous nature.

Protocol for in vitro fluorescence assay of papain-like protease and cell-based immunofluorescence assay of coronavirus infection.

Here, we describe detailed steps to constitute an in vitro assay for monitoring papain-like protease of coronavirus and a cell-based immunofluorescence infection assay. These assays can be adapted for high-throughput screening to determine the efficacy of novel protease inhibitors of coronaviruses and other viruses. In addition, cell-based immunofluorescence infection assay can be used to visually analyze antiviral efficacy of any novel compounds. For complete details on the use and execution of this protocol, please refer to Jeong et al. (2022).1.

Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay.

To identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections.

Chemical screen uncovers novel structural classes of inhibitors of the papain-like protease of coronaviruses.

The papain-like protease (PLpro) of coronaviruses is an attractive antiviral target to inhibit both viral replication and interference of the host immune response. We have identified and characterized three novel classes of small molecules, thiophene, cyanofuran, and triazoloquinazoline, as PLpro inhibitors. Thiophene inhibited the PLpro of two major coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) including SARS-CoV-2, while cyanofuran and triazoloquinazoline more selectively inhibited MERS-CoV PLpro. Unlike GRL0617, a known PLpro inhibitor, all three compounds contain no naphthyl group but like GRL0617 were predicted to fit on the cleft near the BL2 loop. Docking studies further revealed that the location and direction of the binding determined their specificity to different coronaviruses. Together, our work demonstrates that the BL2 loop and nearby regions are outstanding druggable targets, and our three inhibitors can be applicable to the development of therapeutics for coronavirus infection.

A small molecule redistributes iron in ferroportin-deficient mice and patient-derived primary macrophages.

Deficiencies of the transmembrane iron-transporting protein ferroportin (FPN1) cause the iron misdistribution that underlies ferroportin disease, anemia of inflammation, and several other human diseases and conditions. A small molecule natural product, hinokitiol, was recently shown to serve as a surrogate transmembrane iron transporter that can restore hemoglobinization in zebrafish deficient in other iron transporting proteins and can increase gut iron absorption in FPN1-deficient flatiron mice. However, whether hinokitiol can restore normal iron physiology in FPN1-deficient animals or primary cells from patients and the mechanisms underlying such targeted activities remain unknown. Here, we show that hinokitiol redistributes iron from the liver to red blood cells in flatiron mice, thereby increasing hemoglobin and hematocrit. Mechanistic studies confirm that hinokitiol functions as a surrogate transmembrane iron transporter to release iron trapped within liver macrophages, that hinokitiol-Fe complexes transfer iron to transferrin, and that the resulting transferrin-Fe complexes drive red blood cell maturation in a transferrin-receptor-dependent manner. We also show in FPN1-deficient primary macrophages derived from patients with ferroportin disease that hinokitiol moves labile iron from inside to outside cells and decreases intracellular ferritin levels. The mobilization of nonlabile iron is accompanied by reductions in intracellular ferritin, consistent with the activation of regulated ferritin proteolysis. These findings collectively provide foundational support for the translation of small molecule iron transporters into therapies for human diseases caused by iron misdistribution.

Raloxifene prevents intracellular invasion of pathogenic bacteria through modulation of cell metabolic pathways.

BACKGROUND: Pathogenic bacteria can invade and survive inside phagocytic and non-phagocytic cells and use them as a sanctuary against antibiotics. Induction of various host protective mechanisms, including autophagy, can be a novel and effective method to combat intracellular bacteria. Recent studies report that raloxifene, a selective oestrogen receptor modulator, can induce cellular autophagy. OBJECTIVES: To demonstrate the effect of raloxifene on intracellular invasion and proliferation of pathogenic bacteria and investigate raloxifene-induced metabolic changes in host cells. METHODS: Autophagic induction was determined by the extent of nuclear fragmentation and expression levels of the LC3B protein. Intracellular invasion of MRSA strains into A549 lung epithelial cells and invasion of Mycobacterium abscessus into RAW264.7 macrophages were assessed by invasion and proliferation assays. Changes in host cell metabolism were examined by transcriptomic profiling using RNA sequencing. RESULTS: Our data demonstrate increased autophagy in cells upon raloxifene treatment, which contributed to prevention of bacterial invasion and proliferation. Transcriptomic profiling of host cells revealed changes in cholesterol-related pathways and consequent increases in oxidative stress-related genes and in autophagic induction through the TRIM and GABA pathways. CONCLUSIONS: Our results demonstrate raloxifene's potential as a broad-spectrum antibacterial agent through autophagic induction in host cells and prevention of intracellular invasion and proliferation of pathogenic bacteria.

Raloxifene as a treatment option for viral infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused corona virus disease 2019 (COVID-19) pandemic and led to mass casualty. Even though much effort has been put into development of vaccine and treatment methods to combat COVID-19, no safe and efficient cure has been discovered. Drug repurposing or drug repositioning which is a process of investigating pre-existing drug candidates for novel applications outside their original medical indication can speed up the drug development process. Raloxifene is a selective estrogen receptor modulator (SERM) that has been approved by FDA in 1997 for treatment and prevention of postmenopausal osteoporosis and cancer. Recently, raloxifene demonstrates efficacy in treating viral infections by Ebola, influenza A, and hepatitis C viruses and shows potential for drug repurposing for the treatment of SARS-CoV-2 infection. This review will provide an overview of raloxifene's mechanism of action as a SERM and present proposed mechanisms of action in treatment of viral infections.

A pursuit of Staphylococcus aureus continues: a role of persister cells.

Staphylococcus aureus is a pathogen that causes critical diseases, such as pneumonia, endocarditis, and bacteremia, upon gaining access to the bloodstream of the host. Because host innate immunity alone cannot fight against this rapidly expanding pathogen, the use of antibiotic agents is necessary to clear out S. aureus. However, sub-populations of S. aureus fail to respond to the antibiotics resulting in ineffective clearance of the bacteria. One mechanism by which S. aureus does not respond to the antibiotics is by developing resistance through alterations in its genetic makeup, and genetic studies have revealed a major portion of mechanisms that are responsible for the rise of these antibiotic-resistant strains. Another sub-population that fails to respond to the antibiotics is called persister cells. There is a mounting clinical evidence that these persister cells significantly contribute to the antibiotic failure and persistent infection, but a clear mechanistic picture of the formation of the S. aureus persister cells is unavailable. This review focuses on drawing out a mechanistic map of factors that contribute to the formation of S. aureus persister cells. Understanding the mechanism will provide future direction for the development of novel antibiotic strategies to more efficiently tackle infections caused by S. aureus.

Renal clearable nanochelators for iron overload therapy.

Iron chelators have been widely used to remove excess toxic iron from patients with secondary iron overload. However, small molecule-based iron chelators can cause adverse side effects such as infection, gastrointestinal bleeding, kidney failure, and liver fibrosis. Here we report renal clearable nanochelators for iron overload disorders. First, after a singledose intravenous injection, the nanochelator shows favorable pharmacokinetic properties, such as kidney-specific biodistribution and rapid renal excretion (>80% injected dose in 4 h), compared to native deferoxamine (DFO). Second, subcutaneous (SC) administration of nanochelators improves pharmacodynamics, as evidenced by a 7-fold increase in efficiency of urinary iron excretion compared to intravenous injection. Third, daily SC injections of the nanochelator for 5 days to iron overload mice and rats decrease iron levels in serum and liver. Furthermore, the nanochelator significantly reduces kidney damage caused by iron overload without demonstrating DFO's own nephrotoxicity. This renal clearable nanochelator provides enhanced efficacy and safety.

Site-specific intestinal DMT1 silencing to mitigate iron absorption using pH-sensitive multi-compartmental nanoparticulate oral delivery system.

Iron is a nutrient metal, but excess iron promotes tissue damage. Since iron chelation therapies exhibit multiple off-target toxicities, there is a substantial demand for more specific approaches to decrease iron burden in iron overload. While the divalent metal transporter 1 (DMT1) plays a well-established role in the absorption of dietary iron, up-regulation of intestinal DMT1 is associated with iron overload in both humans and rodents. Hence, we developed a novel pH-sensitive multi-compartmental particulate (MCP) oral delivery system that encapsulates DMT1 siRNA and validated its efficacy in mice. Using the gelatin NPs coated with Eudragit® L100-55, we demonstrated that DMT1 siRNA-loaded MCPs down-regulated DMT1 mRNA levels in the duodenum, which was consistent with decreased intestinal absorption of orally-administered 59Fe. Together, the Eudragit® L100-55-based oral siRNA delivery system could provide an effective strategy to specifically down-regulate duodenal DMT1 and mitigate iron absorption.

Brain iron loading impairs DNA methylation and alters GABAergic function in mice.

Iron deficiency is closely associated with altered GABA metabolism and affective behavior. While mutation in the hemochromatosis ( HFE) gene disrupts iron homeostasis and promotes oxidative stress that increases the risk of neurodegeneration, it is largely unknown whether HFE mutation modifies GABAergic homeostasis and emotional behavior. The goal of our study was to investigate the impact of HFE on GABAergic neurochemistry and redox-epigenetic regulation in the brain using H67D HFE-mutant mice that recapitulates the H63D-HFE mutation in humans. H67D mice displayed elevated redox-active iron levels in the brain by 32% compared to age-matched wild-type mice. Moreover, the H67D brain had increased isoprostane and decreased glutathione, indicating elevated oxidative stress. Additionally, the H67D brain had decreased global methylation and attenuated DNA methyltransferase (DNMT) activity. Direct addition of iron to purified DNMT in vitro decreased enzyme activity in a concentration-dependent manner. Last, H67D mice exhibited decreased anxiety-like behavior, which was associated with increased expression of the GABAA receptor α2 subunits by 93%, and these changes were also observed in H67D mice fed a low-iron diet. Taken together, our results suggest a putative role of HFE in regulating labile iron status in the brain, and mutation in H67D perturbs redox-methylation status, contributing to GABAergic dysfunction.-Ye, Q., Trivedi, M., Zhang, Y., Böhlke, M., Alsulimani, H., Chang, J., Maher, T., Deth, R., Kim, J. Brain iron loading impairs DNA methylation and alters GABAergic function in mice.

Iron overload exacerbates age-associated cardiac hypertrophy in a mouse model of hemochromatosis.

Cardiac damage associated with iron overload is the most common cause of morbidity and mortality in patients with hereditary hemochromatosis, but the precise mechanisms leading to disease progression are largely unexplored. Here we investigated the effects of iron overload and age on cardiac hypertrophy using 1-, 5- and 12-month old Hfe-deficient mice, an animal model of hemochromatosis in humans. Cardiac iron levels increased progressively with age, which was exacerbated in Hfe-deficient mice. The heart/body weight ratios were greater in Hfe-deficient mice at 5- and 12-month old, compared with their age-matched wild-type controls. Cardiac hypertrophy in 12-month old Hfe-deficient mice was consistent with decreased alpha myosin and increased beta myosin heavy chains, suggesting an alpha-to-beta conversion with age. This was accompanied by cardiac fibrosis and up-regulation of NFAT-c2, reflecting increased calcineurin/NFAT signaling in myocyte hypertrophy. Moreover, there was an age-dependent increase in the cardiac isoprostane levels in Hfe-deficient mice, indicating elevated oxidative stress. Also, rats fed high-iron diet demonstrated increased heart-to-body weight ratios, alpha myosin heavy chain and cardiac isoprostane levels, suggesting that iron overload promotes oxidative stress and cardiac hypertrophy. Our findings provide a molecular basis for the progression of age-dependent cardiac stress exacerbated by iron overload hemochromatosis.

Role of fatty acid composites in the toxicity of titanium dioxide nanoparticles used in cosmetic products.

It has been recognized that the use of nanoparticles (NPs) in the cosmetic industry results in products with better efficacy and functionality. However, recent advances in molecular toxicology have revealed that NP exposure can promote cytotoxicity and oxidative damage, which has raised health concerns in the use of NPs in personal care products. Nevertheless, the mechanistic basis for the toxicity and safety of cosmetic NPs is poorly understood. The goal of the study was to determine the cytotoxicity and intracellular distribution of titanium dioxide (TiO2) NPs containing fatty acid composites (palmitoleic acid, palmitic acid, stearic acid and oleic acid) commonly used in cosmetic products. Two types of cells, human fibroblast skin cells and adenocarcinoma lung cells, were exposed to either bare TiO2 NPs or TiO2 NPs mixed with fatty acids for up to 48 hr. NMR analysis confirmed that the fatty acid composites remained in the NPs after wash. The cytotoxicity of TiO2 NPs was determined by cell viability measurement using quantitative confocal microscopy, and the localization of two different forms of TiO2 NPs were assessed using electron spectroscopic imaging with transmission electron microscopy. TiO2 NPs containing fatty acids posed significantly reduced cytotoxicity (80-88% decreases) than bare NPs in both cell types. Furthermore, there was less intracellular penetration of the NPs containing fatty acid composites compared with bare NPs. These results provide important insights into the role of fatty acids in protecting the cells from possible toxicity caused by NPs used in the production of cosmetic products.

Loss of divalent metal transporter 1 function promotes brain copper accumulation and increases impulsivity.

The divalent metal transporter 1 (DMT1) is a major iron transporter required for iron absorption and erythropoiesis. Loss of DMT1 function results in microcytic anemia. While iron plays an important role in neural function, the behavioral consequences of DMT1 deficiency are largely unexplored. The goal of this study was to define the neurobehavioral and neurochemical phenotypes of homozygous Belgrade (b/b) rats that carry DMT1 mutation and explore potential mechanisms of these phenotypes. The b/b rats (11-12 weeks old) and their healthy littermate heterozygous (+/b) Belgrade rats were subject to elevated plus maze tasks. The b/b rats spent more time in open arms, entered open arms more frequently and traveled more distance in the maze than +/b controls, suggesting increased impulsivity. Impaired emotional behavior was associated with down-regulation of GABA in the hippocampus in b/b rats. Also, b/b rats showed increased GABAA receptor α1 and GABA transporter, indicating altered GABAergic function. Furthermore, metal analysis revealed that b/b rats have decreased total iron, but normal non-heme iron, in the brain. Interestingly, b/b rats exhibited unusually high copper levels in most brain regions, including striatum and hippocampus. Quantitative PCR analysis showed that both copper importer copper transporter 1 and exporter copper-transporting ATPase 1 were up-regulated in the hippocampus from b/b rats. Finally, b/b rats exhibited increased 8-isoprostane levels and decreased glutathione/glutathione disulfide ratio in the hippocampus, reflecting elevated oxidative stress. Combined, our results suggest that copper loading in DMT1 deficiency could induce oxidative stress and impair GABA metabolism, which promote impulsivity-like behavior. Iron-copper model: Mutations in the divalent metal transporter 1 (DMT1) decrease body iron status and up-regulate copper absorption, which leads to copper loading in the brain and consequently increases metal-induced oxidative stress. This event disrupts GABAergic neurotransmission and promotes impulsivity-like behavior. Our model provides better understanding of physiological risks associated with imbalanced metal metabolism in mental function and, more specifically, the interactions with GABA and redox control in the treatment of emotional disorders.

Short-term manganese inhalation decreases brain dopamine transporter levels without disrupting motor skills in rats.

Manganese (Mn) is used in industrial metal alloys and can be released into the atmosphere during methylcyclopentadienyl manganese tricarbonyl combustion. Increased Mn deposition in the brain after long-term exposure to the metal by inhalation is associated with altered dopamine metabolism and neurobehavioral problems, including impaired motor skills. However, neurotoxic effects of short-term exposure to inhaled Mn are not completely characterized. The purpose of this study is to define the neurobehavioral and neurochemical effects of short-term inhalation exposure to Mn at a high concentration using rats. Male Sprague-Dawley rats were exposed to MnCl2 aerosol in a nose-only inhalation chamber for 3 weeks (1.2 µm, 39 mg/m(3)). Motor coordination was tested on the day after the last exposure using a rotarod device at a fixed speed of 10 rpm for 2 min. Also, dopamine transporter and dopamine receptor protein expression levels in the striatum region of the brain were determined by Western blot analysis. At a rotarod speed of 10 rpm, there were no significant differences in the time on the bar before the first fall or the number of falls during the two-minute test observed in the exposed rats, as compared with controls. The Mn-exposed group had significantly higher Mn levels in the lung, blood, olfactory bulb, prefrontal cortex, striatum, and cerebellum compared with the control group. A Mn concentration gradient was observed from the olfactory bulb to the striatum, supporting the idea that Mn is transported via the olfactory pathway. Our results demonstrated that inhalation exposure to 39 mg/m(3) Mn for 3 weeks induced mild lung injury and modulation of dopamine transporter expression in the brain, without altering motor activity.

Mechanisms of divalent metal toxicity in affective disorders.

Metals are required for proper brain development and play an important role in a number of neurobiological functions. The divalent metal transporter 1 (DMT1) is a major metal transporter involved in the absorption and metabolism of several essential metals like iron and manganese. However, non-essential divalent metals are also transported through this transporter. Therefore, altered expression of DMT1 can modify the absorption of toxic metals and metal-induced toxicity. An accumulating body of evidence has suggested that increased metal stores in the brain are associated with elevated oxidative stress promoted by the ability of metals to catalyze redox reactions, resulting in abnormal neurobehavioral function and the progression of neurodegenerative diseases. Metal overload has also been implicated in impaired emotional behavior, although the underlying mechanisms are not well understood with limited information. The current review focuses on psychiatric dysfunction associated with imbalanced metabolism of metals that are transported by DMT1. The investigations with respect to the toxic effects of metal overload on behavior and their underlying mechanisms of toxicity could provide several new therapeutic targets to treat metal-associated affective disorders.

Influence of lead on repetitive behavior and dopamine metabolism in a mouse model of iron overload.

Exposures to lead (Pb) are associated with neurological problems including psychiatric disorders and impaired learning and memory. Pb can be absorbed by iron transporters, which are up-regulated in hereditary hemochromatosis, an iron overload disorder in which increased iron deposition in various parenchymal organs promote metal-induced oxidative damage. While dysfunction in HFE (High Fe) gene is the major cause of hemochromatosis, the transport and toxicity of Pb in Hfe-related hemochromatosis are largely unknown. To elucidate the relationship between HFE gene dysfunction and Pb absorption, H67D knock-in Hfe-mutant and wild-type mice were given drinking water containing Pb 1.6 mg/ml ad libitum for 6 weeks and examined for behavioral phenotypes using the nestlet-shredding and marble-burying tests. Latency to nestlet-shredding in Pb-treated wild-type mice was prolonged compared with non-exposed wild-types (p < 0.001), whereas Pb exposure did not alter shredding latency in Hfe-mutant mice. In the marble-burying test, Hfe-mutant mice showed an increased number of marbles buried compared with wild-type mice (p = 0.002), indicating more repetitive behavior upon Hfe mutation. Importantly, Pb-exposed wild-type mice buried more marbles than non-exposed wild-types, whereas the number of marbles buried by Hfe-mutant mice did not change whether or not exposed to Pb. These results suggest that Hfe mutation could normalize Pb-induced behavioral alteration. To explore the mechanism of repetitive behavior caused by Pb, western blot analysis was conducted for proteins involved in brain dopamine metabolism. The levels of tyrosine hydroxylase and dopamine transporter increased upon Pb exposure in both genotypes, whereas Hfe-mutant mice displayed down-regulation of the dopamine transporter and dopamine D1 receptor with D2 receptor elevated. Taken together, our data support the idea that both Pb exposure and Hfe mutation increase repetitive behavior in mice and further suggest that these behavioral changes could be associated with altered dopaminergic neurotransmission, providing a therapeutic basis for psychiatric disorders caused by Pb toxicity.