Dopamine­iron homeostasis interaction rescues mitochondrial fitness in Parkinson's disease.

Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.

Design, Synthesis, Electrochemical, and Biological Evaluation of Fluorescent Chlorido[N,N'-bis(methoxy/hydroxy)salicylidene-1,2-bis(4-methoxyphenyl)ethylenediamine]iron(III) Complexes as Anticancer Agents.

The impact of methoxy and hydroxyl groups at the salicylidene moiety of chlorido[N,N'-bis(methoxy/hydroxy)salicylidene-1,2-bis(4-methoxyphenyl)ethylenediamine]iron(III) complexes was evaluated on human MDA-MB 231 breast cancer and HL-60 leukemia cells. Methoxylated complexes (C1-C3) inhibited proliferation, migration, and metabolic activity in a concentration-dependent manner following the rank order: C2 > C3 > C1. In particular, C2 was highly cytotoxic with an IC50 of 4.2 µM which was 6.6-fold lower than that of cisplatin (IC50 of 27.9 µM). In contrast, hydroxylated complexes C4-C6 were almost inactive up to the highest concentration tested due to lack of cellular uptake. C2 caused a dual mode of cell death, ferroptosis, and necroptosis, whereby at higher concentrations, ferroptosis was the preferred form. Ferroptotic morphology and the presence of ferrous iron and lipid reactive oxygen species proved the involvement of ferroptosis. C2 was identified as a promising lead compound for the design of drug candidates inducing ferroptosis.

Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers.

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host's protease profile could affect disease development by changing the toxin's biological features.

Availability of Ferritin-Bound Iron to Enterobacteriaceae.

The sequestration of iron in case of infection, termed nutritional immunity, is an established strategy of host defense. However, the interaction between pathogens and the mammalian iron storage protein ferritin is hitherto not completely understood. To better characterize the function of ferritin in Gram-negative infections, we incubated iron-starved cultures of Salmonella Typhimurium and knockout mutant strains defective for major iron uptake pathways or Escherichia coli with horse spleen ferritin or ionic iron as the sole iron source. Additionally, we added bovine superoxide dismutase and protease inhibitors to the growth medium to assess the effect of superoxide and bacterial proteases, respectively, on Salmonella proliferation and reductive iron release. Compared to free ionic iron, ferritin-bound iron was less available to Salmonella, but was still sufficient to significantly enhance the growth of the bacteria. In the absence of various iron acquisition genes, the availability of ferritin iron further decreased. Supplementation with superoxide dismutase significantly reduced the growth of the ΔentC knockout strain with holoferritin as the sole iron source in comparison with ionic ferrous iron. In contrast, this difference was not observed in the wildtype strain, suggesting that superoxide dismutase undermines bacterial iron uptake from ferritin by siderophore-independent mechanisms. Ferritin seems to diminish iron availability for bacteria in comparison to ionic iron, and its iron sequestering effect could possibly be enhanced by host superoxide dismutase activity.

Breathing, (S)Training and the Pelvic Floor-A Basic Concept.

BACKGROUND: The current scientific literature is inconsistent regarding the potential beneficial or deleterious effects of high-intensity physical activities on the pelvic floor (PF) in women. So far, it has not been established with certainty whether disparate breathing mechanisms may exert short- or long-term influence on the PF function in this context, although based on the established physiological interrelationship of breathing with PF activation, this seems plausible. OBJECTIVE: To propose a basic concept of the influence of different breathing patterns on the PF during strenuous physical efforts. Methodical approaches: Review of the recent literature, basic knowledge of classical western medicine regarding the principles of muscle physiology and the biomechanics of breathing, additional schematic illustrations, and magnetic resonance imaging (MRI) data corroborate the proposed concept and exemplify the consequences of strenuous efforts on the PF in relation to respective breathing phases. CONCLUSION: The pelvic floor muscles (PFMs) physiologically act as expiratory muscles in synergy with the anterolateral abdominal muscles, contracting during expiration and relaxing during inspiration. Obviously, a strenuous physical effort requires an expiratory motor synergy with the PFM and abdominal muscles in a co-contracted status to train the PFM and protect the PF against high intra-abdominal pressure (IAP). Holding breath in an inspiratory pattern during exertion stresses the PF because the high IAP impinges on the relaxed, hence insufficiently protected, PFMs. It seems conceivable that such disadvantageous breathing, if performed regularly and repeatedly, may ultimately cause PF dysfunction. At any rate, future research needs to take into account the respective breathing cycles during measurements and interventions addressing PFM function.

Ambient Availability of Amino Acids, Proteins, and Iron Impacts Copper Resistance of Aspergillus fumigatus.

The transition metals iron and copper are required by virtually all organisms but are toxic in excess. Acquisition of both metals and resistance to copper excess have previously been shown to be important for virulence of the most common airborne human mold pathogen, Aspergillus fumigatus. Here we demonstrate that the ambient availability of amino acids and proteins increases the copper resistance of A. fumigatus wild type and particularly of the ΔcrpA mutant that lacks export-mediated copper detoxification. The highest-protecting activity was found for L-histidine followed by L-asparagine, L-aspartate, L-serine, L-threonine, and L-tyrosine. Other amino acids and proteins also displayed significant but lower protection. The protecting activity of non-proteinogenic D-histidine, L-histidine-mediated growth inhibition in the absence of high-affinity copper uptake, determination of cellular metal contents, and expression analysis of copper-regulated genes suggested that histidine inhibits low-affinity but not high-affinity copper acquisition by extracellular copper complexation. An increase in the cellular copper content was found to be accompanied by an increase in the iron content, and, in agreement, iron starvation increased copper susceptibility, which underlines the importance of cellular metal balancing. Due to the role of iron and copper in nutritional immunity, these findings are likely to play an important role in the host niche.

Intracerebral Iron Accumulation may be Associated with Secondary Brain Injury in Patients with Poor Grade Subarachnoid Hemorrhage.

BACKGROUND: The amount of intracranial blood is a strong predictor of poor outcome after subarachnoid hemorrhage (SAH). Here, we aimed to measure iron concentrations in the cerebral white matter, using the cerebral microdialysis (CMD) technique, and to associate iron levels with the local metabolic profile, complications, and functional outcome. METHODS: For the observational cohort study, 36 patients with consecutive poor grade SAH (Hunt & Hess grade of 4 or 5, Glasgow Coma Scale Score ≤ 8) undergoing multimodal neuromonitoring were analyzed for brain metabolic changes, including CMD iron levels quantified by graphite furnace atomic absorption spectrometry. The study time encompassed 14 days after admission. Statistical analysis was performed using generalized estimating equations. RESULTS: Patients were admitted in a poor clinical grade (n = 26, 72%) or deteriorated within 24 h (n = 10, 28%). The median blood volume in the subarachnoid space was high (SAH sum score = 26, interquartile range 20-28). Initial CMD iron was 44 µg/L (25-65 µg/L), which significantly decreased to a level of 25 µg/L (14-30 µg/L) at day 4 and then constantly increased over the remaining neuromonitoring days (p < 0.01). A higher intraventricular hemorrhage sum score (≥ 5) was associated with higher CMD iron levels (Wald-statistic = 4.1, df = 1, p = 0.04) but not with the hemorrhage load in the subarachnoid space (p = 0.8). In patients developing vasospasm, the CMD iron load was higher, compared with patients without vasospasm (Wald-statistic = 4.1, degree of freedom = 1, p = 0.04), which was not true for delayed cerebral infarction (p = 0.4). Higher iron concentrations in the brain extracellular fluid (34 µg/L, 36-56 µg/L vs. 23 µg/L, 15-37 µg/L) were associated with mitochondrial dysfunction (CMD lactate to pyruvate ratio > 30 and CMD-pyruvate > 70 µM/L, p < 0.001). Brain extracellular iron load was not associated with functional outcome after 3 months (p > 0.5). CONCLUSIONS: This study suggests that iron accumulates in the cerebral white matter in patients with poor grade SAH. These findings may support trials aiming to scavenger brain extracellular iron based on the hypothesis that iron-mediated neurotoxicity may contribute to acute and secondary brain injury following SAH.

Cell-specific expression of Hfe determines the outcome of Salmonella enterica serovar Typhimurium infection in mice

Mutations in HFE cause hereditary hemochromatosis type I hallmarked by increased iron absorption, iron accumulation in hepatocytes and iron deficiency in myeloid cells. HFE encodes an MHC-I like molecule, but its function in immune responses to infection remains incompletely understood. Here, we investigated putative roles of Hfe in myeloid cells and hepatocytes, separately, upon infection with Salmonella Typhimurium, an intracellular bacterium with iron-dependent virulence. We found that constitutive and macrophage-specific deletion of Hfe protected infected mice. The propagation of Salmonella in macrophages was reduced due to limited intramacrophage iron availability for bacterial growth and increased expression of the anti-microbial enzyme nitric oxide synthase-2. By contrast, mice with hepatocyte-specific deletion of Hfe succumbed earlier to Salmonella infection because of unrestricted extracellular bacterial replication associated with high iron availability in the serum and impaired expression of essential host defense molecules such as interleukin-6, interferon-γ and nitric oxide synthase-2. Wild-type mice subjected to dietary iron overload phenocopied hepatocyte-specific Hfe deficiency suggesting that increased iron availability in the serum is deleterious in Salmonella infection and underlies impaired host immune responses. Moreover, the macrophage-specific effect is dominant over hepatocyte-specific Hfe-depletion, as Hfe knock-out mice have increased survival despite the higher parenchymal iron load associated with systemic loss of Hfe. We conclude that cell-specific expression of Hfe in hepatocytes and macrophages differentially affects the course of infections with specific pathogens by determining bacterial iron access and the efficacy of anti-microbial immune effector pathways. This may explain the high frequency and evolutionary conservation of human HFE mutations.

Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines.

Enterohemorrhagic Escherichia coli (EHEC) infections can cause EHEC-associated hemolytic uremic syndrome (eHUS) via its main virulent factor, Shiga toxins (Stxs). Complement has been reported to be involved in the progression of eHUS. The aim of this study was to investigate the interactions of the most effective subtype of the toxin, Stx2a, with pivotal complement proteins C3b and C5. The study further examined the effect of Stx2a stimulation on the transcription and synthesis of these complement proteins in human target cell lines. Binding of Stx2a to C3b and C5 was evaluated by ELISA. Kidney and gut cell lines (HK-2 and HCT-8) were stimulated with varied concentrations of Stx2a. Subsequent evaluation of complement gene transcription was studied by real-time PCR (qPCR), and ELISAs and Western blots were performed to examine protein synthesis of C3 and C5 in supernatants and lysates of stimulated HK-2 cells. Stx2a showed a specific binding to C3b and C5. Gene transcription of C3 and C5 was upregulated with increasing concentrations of Stx2a in both cell lines, but protein synthesis was not. This study demonstrates the binding of Stx2a to complement proteins C3b and C5, which could potentially be involved in regulating complement during eHUS infection, supporting further investigations into elucidating the role of complement in eHUS pathogenesis.

The structure of the Shiga toxin 2a A-subunit dictates the interactions of the toxin with blood components.

Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)-producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A-subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities. Uncleaved Stx2a was confirmed to bind to human neutrophils and to trigger leukocyte/platelet aggregate formation, whereas cleaved Stx2a was ineffective. Conversely, binding of complement factor H was confirmed for cleaved Stx2a and not for uncleaved Stx2a. It is worth noting that uncleaved and cleaved Stx2a showed no differences in cytotoxicity for Vero cells or Raji cells, structural conformation, and contaminating endotoxin. These results have been obtained by comparing two Stx2a batches, purified in different laboratories by using different protocols, termed Stx2a(cl; cleaved toxin, Innsbruck) and Stx2a(uncl; uncleaved toxin, Bologna). Stx2a(uncl) behaved as Stx2a(cl) after mild trypsin treatment. In this light, previous controversial results obtained with purified Stx2a has to be critically re-evaluated; furthermore, characterisation of the structure of circulating Stx2a is mandatory to understand eHUS-pathogenesis and to develop therapeutic approaches.

Shiga toxin 2a binds antithrombin and heparin, but does not directly activate platelets.

Escherichia coli-induced hemolytic uremic syndrome (eHUS) is a life-threatening complication of infection with Shiga toxin (Stx), in particular Stx2a-producing Escherichia coli. Enhanced coagulation activation with formation of microthrombi seems to be a key event in development of eHUS. Platelet activation has been postulated as a possible, but controversially debated mechanism. The present study investigated the effect of Stx2a on plasmatic coagulation and platelets. Binding studies were initially performed with ELISA and co-immunoprecipitation and supported by quartz crystal microbalance with dissipation monitoring (QCM-D). Antithrombin (AT) activity was measured using the automated BCS XP® system. ROTEM® was used for functional coagulation testing. Platelet binding and activation was studied with FACS and light-transmission aggregometry. We found binding of Stx2a to AT, an important inhibitor of blood coagulation, but only a mild albeit significant reduction of AT activity against FXa in the presence of Stx2a. QCM-D analysis also showed binding of Stx2a to heparin and an impaired binding of AT to Stx2a-bound heparin. ROTEM® using Stx2a-treated platelet-poor plasma revealed a significant, but only moderate shortening of clotting time. Neither binding nor activation of platelets by Stx2a could be demonstrated. In summary, data of this study suggest that Stx2a binds to AT, but does not induce major effects on plasmatic coagulation. In addition, no interaction with platelets occurred. The well-known non-beneficial administration of heparin in eHUS patients could be explained by the interaction of Stx2a with heparin.

Comparing the effect of strontium-functionalized and fluoride-modified surfaces on early osseointegration.

BACKGROUND: Studies have shown that medical devices comprising strontium contribute to bone healing and osseointegration. The aim of this study was to evaluate the in vivo performance of surface-functionalized implants (Ti-Sr-O) showing predictable release characteristics of strontium and compare it to performance a commercially available fluoride-modified surface. METHODS: Ti-Sr-O functionalized, fluoride-modified,  Grade 4 titanium implants were inserted in the femoral condyle of adult male New Zealand white rabbits. Atomic absorption spectrometry (AAS) was utilized to monitor strontium blood serum levels. Two weeks after insertion, histomorphometric evaluation was performed with respect to bone-to-implant contact (BIC%) and bone formation (BF%) using defined regions of interest. RESULTS: Mean values for BIC% showed a comparable degree of osseointegration for Ti-Sr-O and the fluoride-modified surface, while BF% revealed a significant difference in increased BF with Ti-Sr-O. AAS measurements did not indicate any influence of the Ti-Sr-O modified implants on the strontium blood serum concentrations. CONCLUSIONS: Within the limitations of this study, it was shown that the Ti-Sr-O coating, with sustained release characteristics of strontium, enhanced bone apposition and, thus, could find practical applications, e.g., within the field of medical implantology.

Effect of strontium surface-functionalized implants on early and late osseointegration: A histological, spectrometric and tomographic evaluation.

Numerous in vivo, in vitro and clinical studies report on beneficial effects of strontium with respect to increased bone growth. Based on this knowledge the aim of this study was to evaluate early and late osseointegration stages of functionalized titanium implants showing sustained release of strontium (Sr) and further investigate its potential systemic effect. Strontium functionalized (Ti-Sr-O) and Grade 4 (Control) titanium implants were inserted in the femoral condyle of New Zealand White rabbits. The Ti-Sr-O coating was characterized using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectrometry (EDX) for structure, coating thickness and chemical composition. Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) was used to evaluate released strontium in vitro while Atomic Absorption Spectrometry (AAS) was utilized to monitor serum levels of strontium and calcium. Additionally, histological and tomographic analysis of bone-to-implant contact (BIC%) and bone formation (BF%) was performed, following implantation periods of two or twelve weeks, respectively. Median values for BIC% for Ti-Sr-O revealed significant differences within the two- and twelve-week observation periods, while exceeding BF% was discovered especially after twelve weeks when performing the histological evaluation. The results from the micro-computed tomography (µ-CT) showed no significant differences, when comparing the experimental groups. AAS measurements did not indicate a systemic effect by the local strontium release. Within the limitations of the study, it was shown that a Ti-Sr-O coating with sustained release characteristics of strontium, accelerates bone apposition and represents a potential potent surface modification for endosseous medical implant devices. STATEMENT OF SIGNIFICANCE: This study presents first data with respect to early and late in vivo response on a strontium functionalized titanium surface comprising a nanotopography manufactured by a magnetron sputtering process. We investigated different osseointegration stages of screw-shaped implants with dental implant geometries in a rabbit femur model observing beneficial effects of the functionalized surface on bone-to-implant contact and bone formation caused by tailored release of the bone anabolic strontium. Histomorphometrical data revealed that a functionalized titanium surface with controlled liberation of strontium accelerates osseointegration while spectrometry measurements did not indicate a potential systemic effect of this osteoinductive agent and could thus have impact on modifications of medical implant devices.

Salmonella Utilizes Zinc To Subvert Antimicrobial Host Defense of Macrophages via Modulation of NF-κB Signaling.

Zinc sequestration by macrophages is considered a crucial host defense strategy against infection by the intracellular bacterium Salmonella enterica serovar Typhimurium. However, the underlying mechanisms remain elusive. In this study, we found that zinc favors pathogen survival within macrophages. Salmonella-hosting macrophages contained higher free zinc levels than did uninfected macrophages and cells that successfully eliminated bacteria, which was paralleled by the impaired production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in bacterium-harboring cells. A profound, zinc-mediated inhibition of NF-κB p65 transcriptional activity affecting the expression of the ROS- and RNS-forming enzymes phos47 and inducible nitric oxide synthase (iNOS) provided a mechanistic explanation for this phenomenon. Macrophages responded to infection by enhancing the expression of zinc-scavenging metallothioneins 1 and 2, whose genetic deletion caused increased free zinc levels, reduced ROS and RNS production, and increased the survival of Salmonella Our data suggest that Salmonella invasion of macrophages results in a bacterium-driven increase in the intracellular zinc level, which weakens antimicrobial defense and the ability of macrophages to eradicate the pathogen. Thus, limitation of cytoplasmic zinc levels may help to control infection by intracellular bacteria.

Use of Underarm Cosmetic Products in Relation to Risk of Breast Cancer: A Case-Control Study.

BACKGROUND: Previous studies on breast cancer (BC), underarm cosmetic products (UCP) and aluminum salts have shown conflicting results. We conducted a 1:1 age-matched case-control study to investigate the risk for BC in relation to self-reported UCP application. METHODS: Self-reported history of UCP use was compared between 209 female BC patients (cases) and 209 healthy controls. Aluminum concentration in breast tissue was measured in 100 cases and 52 controls. Multivariable conditional logistic regression analysis was performed to estimate odds ratios (ORs) with 95% confidence intervals (CIs), adjusting for established BC risk factors. FINDINGS: Use of UCP was significantly associated with risk of BC (p=0.036). The risk for BC increased by an OR of 3.88 (95% CI 1.03-14.66) in women who reported using UCP's several times daily starting at an age earlier than 30years. Aluminum in breast tissue was found in both cases and controls and was significantly associated to self-reported UCP use (p=0.009). Median (interquartile) aluminum concentrations were significantly higher (p=0.001) in cases than in controls (5.8, 2.3-12.9 versus 3.8, 2.5-5.8nmol/g). INTERPRETATION: Frequent use of UCPs may lead to an accumulation of aluminum in breast tissue. More than daily use of UCPs at younger ages may increase the risk of BC.

Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells.

Altered mitochondrial metabolism plays a pivotal role in the development and progression of various diseases, including cancer. Cell lines are frequently used as models to study mitochondrial (dys)function, but little is known about their mitochondrial respiration and metabolic properties in comparison to the primary tissue of origin. We have developed a method for assessment of oxidative phosphorylation in prostate tissue samples of only 2 mg wet weight using high-resolution respirometry. Reliable protocols were established to investigate the respiratory activity of different segments of the mitochondrial electron transfer system (ETS) in mechanically permeabilized tissue biopsies. Additionally, the widely used immortalized prostate epithelial and fibroblast cell lines, RWPE1 and NAF, representing the major cell types in prostate tissue, were analyzed and compared to the tissue of origin. Our results show that mechanical treatment without chemical permeabilization agents or sample processing constitutes a reliable preparation method for OXPHOS analysis in small amounts of prostatic tissue typically obtained by prostate biopsy. The cell lines represented the bioenergetic properties of fresh tissue to a limited extent only. Particularly, tissue showed a higher oxidative capacity with succinate and glutamate, whereas pyruvate was a substrate supporting significantly higher respiratory activities in cell lines. Several fold higher zinc levels measured in tissue compared to cells confirmed the role of aconitase for prostate-specific metabolism in agreement with observed respiratory properties. In conclusion, combining the flexibility of cell culture models and tissue samples for respirometric analysis are powerful tools for investigation of mitochondrial function and tissue-specific metabolism.

Heme oxygenase 1 controls early innate immune response of macrophages to Salmonella Typhimurium infection.

Macrophages are central for the immune control of intracellular microbes. Heme oxygenase 1 (HO-1, hmox) is the first and rate limiting enzyme in the breakdown of heme originating from degraded senescent erythrocytes and heme-proteins, yielding equal amounts of iron, carbon monoxide and biliverdin. HO-1 is strongly up-regulated in macrophages in response to inflammatory signals, including bacterial endotoxin. In view of the essential role of iron for the growth and proliferation of intracellular bacteria along with known effects of the metal on innate immune function, we examined whether HO-1 plays a role in the control of infection with the intracellular bacterium Salmonella Typhimurium. We studied the course of infection in stably-transfected murine macrophages (RAW264.7) bearing a tetracycline-inducible plasmid producing hmox shRNA and in primary HO-1 knockout macrophages. While uptake of bacteria into macrophages was not affected, a significantly reduced survival of intracellular Salmonella was observed upon hmox knockdown or pharmacological hmox inhibition, which was independent of Nramp1 functionality. This could be traced to limitation of iron availability for intramacrophage bacteria along with enhanced stimulation of innate immune effector pathways, including the formation of reactive oxygen and nitrogen species and increased TNF-α expression. Mechanistically, these latter effects result from intracellular iron limitation with subsequent activation of NF-κB and further inos, tnfa and p47phox transcription along with reduced formation of the anti-inflammatory and radical scavenging molecules, CO and biliverdin as a consequence of HO-1 silencing. Taken together our data provide novel evidence that the infection-driven induction of HO-1 exerts detrimental effects in the early control of Salmonella infection, whereas hmox inhibition can favourably modulate anti-bacterial immune effector pathways of macrophages and promote bacterial elimination.

Lipocalin-2 ensures host defense against Salmonella Typhimurium by controlling macrophage iron homeostasis and immune response.

Lipocalin-2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron-laden bacterial siderophores, chemo-attracts neutrophils and has immunomodulatory and apoptosis-regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella-infected macrophages, which reduces cellular iron content and enhances the generation of pro-inflammatory cytokines. Lcn2 represses IL-10 production while augmenting Nos2, TNF-α, and IL-6 expression. Lcn2(-/-) macrophages have elevated IL-10 levels as a consequence of increased iron content. The crucial role of Lcn-2/IL-10 interactions was further demonstrated by the greater ability of Lcn2(-/-) IL-10(-/-) macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2(-/-) counterparts. Overexpression of the iron exporter ferroportin-1 in Lcn2(-/-) macrophages represses IL-10 and restores TNF-α and IL-6 production to the levels found in wild-type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL-10 production, and that both functions are linked to its ability to shuttle iron from macrophages.

Characterization of the Link between Ornithine, Arginine, Polyamine and Siderophore Metabolism in Aspergillus fumigatus.

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.