Nramp1 Expression Research Articles

The expression of Nramp1 modulates the uptake of Mycobacterium tuberculosis by macrophages through alternating inflammatory responses

Natural-resistance-associated macrophage protein-1 (NRAMP1) is a transmembrane protein of the mammalian SLC11 gene family. Previously, genome-wide association study (GWAS) have shown that the single nucleotide polymorphisms (SNPs) of NRAMP1 are associated with human susceptibility to tuberculosis (TB), and the detection of clinical samples have demonstrated that the expression levels of NRAMP1 are concomitant with the susceptibility to TB in humans and cows, but underlying mechanism is unknown. In this study, we completed a series of experiments to investigate how the expression of Nramp1 affects the infection of macrophages with Mycobacterium tuberculosis (Mtb). We found that the increase of Nramp1 expression induced the decrease of Mtb infection efficiency and the higher-level expression of pro-inflammatory cytokines and chemokines, However, the knockdown of Nramp1 promoted the efficiency of bacilli infection to macrophages and induced lower-levels of expression of pro-inflammatory cytokines and chemokines. Collectively, the results in this study demonstrated that the levels of Nramp1 expression affect Mtb infection of macrophage and regulate pro-inflammatory responses of macrophages to Mtb infection, indicating the population with the low-expression level of NRAMP1 predispose to Mtb infection and TB development, and suggesting SNPs in NRAMP1 modulate the host susceptibility to TB through its regulation of NRAMP1 expression.

Localized Leishmania major infection disrupts systemic iron homeostasis that can be controlled by oral iron supplementation

Leishmania parasites are heavily dependent on efficient iron acquisition from a tightly regulated host iron pool for survival and virulence. Prior studies uncovered multiple strategies adopted by the parasite to hijack the iron-regulatory network of macrophages. Despite these extensive studies with infected macrophages, there is limited knowledge of the effect of Leishmania infection on systemic iron homeostasis. This issue is particularly relevant for Leishmania major, which causes localized skin infection with minimal lymphatic spread. We show for the first time that L. major infection in the mouse footpad induced influx of iron at the site of infection through blood with simultaneous upregulation of transferrin receptor 1 (TfR1) and downregulation of phagolysosomal iron exporter Nramp1 expression in the footpad tissue. Interestingly, localized L. major infection had far-reaching effects beyond the infection site triggering anemia-like symptoms. This was evident from depleted physiological iron stores from the liver and bone marrow as well as reduced hemoglobin levels and deformed erythrocytes. The infected mice also developed splenomegaly with signs of splenic stress erythropoiesis as indicated by upregulation of several erythroid-related genes. These observations prompted us to provide oral iron supplementations to the L. major infected mice, which resulted in a drastic reduction of the parasite load and restoration of iron homeostasis.

Citric acid assisted phytoextraction of nickle from soil helps to tolerate oxidative stress and expression profile of NRAMP genes in sunflower at different growth stages.

Soil polluted with Nickel (Ni) adversely affects sunflower growth resulting in reduced yield. Counterbalancing Ni toxicity requires complex molecular, biochemical, and physiological mechanisms at the cellular, tissue, and whole plant levels, which might improve crop productivity. One of the primary adaptations to tolerate Ni toxicity is the enhanced production of antioxidant enzymes and the elevated expression of Ni responsive genes. In this study, biochemical parameters, production of ROS, antioxidants regulation, and expression of NRAMP metal transporter genes were studied under Ni stress in sunflower. There were four soil Ni treatments (0, 50, 100, and 200 mg kg-1 soil), while citric acid (CA, 5 mM kg-1 soil) was applied on the 28th and 58th days of plant growth. The samples for all analyses were obtained on the 30th and 60th day of plant growth, respectively. The results indicated that the concentrations of Ni in roots and shoots were increased with increasing concentrations of Ni at both time intervals. Proline contents, ascorbic acid, protein, and total phenolics were reduced under Ni-stress, but with the application of CA, improvement was witnessed in their contents. The levels of malondialdehyde and hydrogen peroxide were enhanced with the increasing concentration of Ni, and after applying CA, they were reduced. The contents of antioxidants, i.e., catalase, peroxidase, superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase, were increased at 50 ppm Ni concentration and decreased at higher concentrations of Ni. The application of CA significantly improved antioxidants at all concentrations of Ni. The enhanced expression of NRAMP1 (4, 51 and 81 folds) and NRAMP3 (1.05, 4 and 6 folds) was found at 50, 100 and 200ppm Ni-stress, respectively in 30 days old plants and the same pattern of expression was recorded in 60 days old plants. CA further enhanced the expression at both developmental stages. In conclusion, CA enhances Ni phytoextraction efficiency as well as protect plant against oxidative stress caused by Ni in sunflower.

Fe and Zn stress induced gene expression analysis unraveled mechanisms of mineral homeostasis in common bean (Phaseolus vulgaris L.)

Iron (Fe) and zinc (Zn) stress significantly affects fundamental metabolic and physiological processes in plants that results in reduction of plant growth and development. In the present study, common bean variety; Shalimar French Bean-1 (SFB-1) was used as an experimental material. Four different MGRL media i.e. normal MGRL medium (Control), media without Fe (0-Fe), media without Zn (0-Zn) and media with excess Zn (300-Zn) were used for growing seeds of SFB-1 under in vitro condition for three weeks under optimum conditions. Three week old shoot and root tissues were harvested from the plants grown in these four different in vitro conditions and were, subjected to Fe and Zn estimation. Further, extraction of total RNA for differential gene expression of ten candidate genes selected based on our in silico investigation and their classification, phylogeny and expression pattern was unraveled. Expression analysis of three candidate genes (OPT3, NRAMP2 and NRAMP3) in roots revealed possible cross talk among Fe/Zn stress that was further confirmed by observing less accumulation of Fe in roots under both these conditions. However, we observed, higher accumulation of Fe in shoots under 0-Fe condition compared to control that suggests precise sensing for priority based compartmentalization and partitioning leading to higher accumulation of Fe in shoots. Furthermore, the expression analysis of IRT1, FRO1 and Ferritin 1 genes under Fe/Zn stress suggested their role in uptake/transport and signaling of Fe and Zn, whereas the expression of ZIP2, NRAMP1, HA2 and GLP1 genes were highly responsive to Zn in Phaseolus vulgaris. The identified genes highly responsive to Fe and Zn stress condition can be potential candidates for overcoming mineral stress in dicot crop plants.

Foliar application of gibberellin inhibits the cadmium uptake and xylem transport in lettuce (Lactuca sativa L.)

Gibberellin (GA) has been shown to exert numerous beneficial physiological functions in the resistance of plant to various abiotic stresses, however, the mechanism of GA for the mitigation of Cd stress in vegetables has not been well established. This study was carried out to investigate the effect of GA on the growth and Cd uptake in lettuce plants. Result showed that 10 µmol/L GA treatment restored part of the root morphology and vigor of lettuce under Cd stress by increasing the total root length, surface area and total root tip number, which directly showed an increased biomass of lettuce. On the other hand, GA reduced Cd uptake by roots and Cd root-to-shoot translocation by down-regulated expression of IRT1, Nramp1, HMA2 and HMA4, which finally alleviated the toxic effects of Cd on lettuce leaves, as confirmed by Non-invasive micro-test Technique (NMT) results. Overall, these findings provide new insights into the mechanism of cadmium toxicity mitigation in vegetables by GA and provide a theoretical basis for the development of physiological blockers with gibberellin components.

Leishmania infection triggers hepcidin-mediated proteasomal degradation of Nramp1 to increase phagolysosomal iron availability.

Natural resistance-associated macrophage protein 1 (Nramp1) was originally discovered as a genetic determinant of resistance against multiple intracellular pathogens, including Leishmania. It encodes a transmembrane protein of the phago-endosomal compartments, where it functions as an iron transporter. But the mechanism by which Nramp1 controls host-pathogen dynamics and determines final outcome of an infection is yet to be fully deciphered. Whether the expression of Nramp1 is altered in response to a pathogen attack is also unknown. To address these, Nramp1 status was examined in Leishmania major-infected murine macrophages. We observed that at 12 hrs post infection, there was drastic lowering of Nramp1 level accompanied by increased phagolysosomal iron content and enhanced intracellular parasite growth. Leishmania infection-induced Nramp1 downregulation was caused by ubiquitin-proteasome degradation pathway, which in turn was found to be mediated by the iron-regulatory peptide hormone hepcidin. Blocking of Nramp1 degradation with proteasome inhibitor or transcriptional agonist of hepcidin resulted in depletion of phagolysosomal iron pool that led to significant reduction of intracellular parasite burden. Interestingly, Nramp1 level was restored to normalcy after 30 hrs of infection with a concomitant drop in phagolysosomal iron, which is suggestive of a host counteractive response to deprive the pathogen of this essential micronutrient. Taken together, our study implicates Nramp1 as a central player in the host-pathogen battle for phagolysosomal iron. We also report Nramp1 as a novel target for hepcidin, and this 'hepcidin-Nramp1' axis may have a broader role in regulating macrophage iron homeostasis.

Abscisic acid (ABA)-importing transporter 1 (AIT1) contributes to the inhibition of Cd accumulation via exogenous ABA application in Arabidopsis

Soil cadmium (Cd) accumulation presents risks to crop safety and productivity. However, through an exogenous application of abscisic acid (ABA), its accumulation in plants can be reduced and its toxicity mitigated, thereby providing an alternative strategy to counteract Cd contamination of arable soil. In the present study, we demonstrated that exogenous ABA application alleviates Cd-induced growth inhibition and photosynthetic damage in wild-type (Col-0) Arabidopsis plants. However, these positive effects were weakened in the ABA-importing transporter (AIT1)-deficient mutant (ait1). Through further analysis, we found that upon ABA application, the decrease in Cd level significantly differed among ait1, Col-0, and the two AIT1-overexpressing transgenic plants (AIT1ox-1 and AIT1ox-2), suggesting that AIT1 mediates the Cd-reducing effects of ABA. ABA application also inhibited the expression of IRT1, ZIP1, ZIP4, and Nramp1 in Col-0 plants subjected to Cd stress. However, significant differences among the genotypes (ait1, Col-0 and AIT1ox) were only observed in terms of IRT1 expression. Overall, our findings suggest that the suppression of Cd accumulation and restoration of plant growth by exogenous ABA require the ABA-importing activity of AIT1 to inhibit IRT1 expression.

Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter

Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter

Expression kinetics of natural resistance associated macrophage protein (NRAMP) genes in Salmonella Typhimurium-infected chicken

BackgroundSalmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a zoonotic pathogen responsible for severe intestinal pathology in young chickens. Natural resistance-associated macrophage protein (NRAMP) family has been shown to be associated with resistance to intracellular pathogens, including Salmonella Typhimurium. The role of NRAMP proteins in macrophage defence against microbial infection has been ascribed to changes in the metal-ion concentrations inside the bacteria-containing phagosomes. The present study was conducted to investigate tissue-specific (liver, spleen and caecum) expression kinetics of NRAMP gene family (NRAMP1 and NRAMP2) in broilers from day 0 to day 15 after Salmonella Typhimurium challenge concomitant to clinical, blood biochemical and immunological parameters survey.ResultsClinical symptoms appeared 4 days post-infection (dpi) in infected birds. Symptoms like progressive weakness, anorexia, diarrhoea and lowering of the head were seen in infected birds one-week post-infection. On postmortem examination, liver showed congestion, haemorrhage and necrotic foci on the surface, while as the spleen, lungs and intestines revealed congestion and haemorrhages. Histopathological alterations were principally found in liver comprising of necrosis, reticular endothelial hyperplasia along with mononuclear cell and heterophilic infiltration. Red Blood Cell (RBC) count, Haemoglobin (Hb) and Packed Cell Volume (PCV) decreased significantly (P < 0.05) in blood while heterophil counts increased up to 7 days post-infection. Serum glucose, aspartate transaminase (AST) and alanine transaminase (ALT) enzymes concentrations increased significantly throughout the study. A gradual increase of specific humoral IgG response confirmed Salmonella infection. Meanwhile, expression of NRAMP1 and NRAMP2 genes was differentially regulated after infection in tissues such as liver, spleen and caecum known to be the target of Salmonella Typhimurium replication in the chicken.ConclusionThus the specific roles of NRAMP1 and NRAMP2 genes in Salmonella Typhimurium induced disease may be supposed from their differential expression according to tissues and timing after per os infection. However, these roles remain to be analyzed related to the severity of the disease which can be estimated by blood biochemistry and immunological parameters.

Down-regulation of natural resistance-associated macrophage protein-1 (Nramp1) is associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ )-induced α-synuclein accumulation and neurotoxicity.

The accumulation of α-synuclein is a hallmark in the pathogenesis of Parkinson's disease (PD). Natural resistance-associated macrophage protein-1 (Nramp1) was previously shown to contribute to the degradation of extracellular α-synuclein in microglia under conditions of iron overload. This study was aimed at investigating the role of Nramp1 in α-synuclein pathology in the neurone under 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ ) treatment. The expression of Nramp1 and pathological features (including iron and α-synuclein accumulation) were examined in the dopaminergic neurones of humans (with and without PD) and of mice [with and without receiving chronic MPTP intoxication]. The effects of Nramp1 expression on low-dose MPP+ -induced α-synuclein expression and neurotoxicity were determined in human dopaminergic neuroblastoma SH-SY5Y cells. Similar to the findings in the substantia nigra of human PD, lower expression of Nramp1 but higher levels of iron and α-synuclein were identified in the dopaminergic neurones of mice receiving chronic MPTP intoxication, compared to controls. In parallel to the loss of dopaminergic neurones, the numbers of glial fibrillary acidic protein- and ionized calcium-binding adapter molecule-1-positive cells were significantly increased in the substantia nigra of MPTP-treated mice. Likewise, in human neuroblastoma SH-SY5Y cells exposed to low-dose MPP+ , Nramp1 expression and cathepsin D activity were decreased, along with an increase in α-synuclein protein expression and aggregation. Overexpression of functional Nramp1 restored cathepsin D activity and attenuated α-synuclein up-regulation and neuronal cell death caused by MPP+ treatment. These data suggest that the neuronal expression of Nramp1 is important for protecting against the development of MPTP/MPP+ -induced α-synuclein pathology and neurotoxicity.

Developmental Control of NRAMP1 (SLC11A1) Expression in Professional Phagocytes.

NRAMP1 (SLC11A1) is a professional phagocyte membrane importer of divalent metals that contributes to iron recycling at homeostasis and to nutritional immunity against infection. Analyses of data generated by several consortia and additional studies were integrated to hypothesize mechanisms restricting NRAMP1 expression to mature phagocytes. Results from various epigenetic and transcriptomic approaches were collected for mesodermal and hematopoietic cell types and compiled for combined analysis with results of genetic studies associating single nucleotide polymorphisms (SNPs) with variations in NRAMP1 expression (eQTLs). Analyses establish that NRAMP1 is part of an autonomous topologically associated domain delimited by ubiquitous CCCTC-binding factor (CTCF) sites. NRAMP1 locus contains five regulatory regions: a predicted super-enhancer (S-E) key to phagocyte-specific expression; the proximal promoter; two intronic areas, including 3′ inhibitory elements that restrict expression during development; and a block of upstream sites possibly extending the S-E domain. Also the downstream region adjacent to the 3′ CTCF locus boundary may regulate expression during hematopoiesis. Mobilization of the locus 14 predicted transcriptional regulatory elements occurs in three steps, beginning with hematopoiesis; at the onset of myelopoiesis and through myelo-monocytic differentiation. Basal expression level in mature phagocytes is further influenced by genetic variation, tissue environment, and in response to infections that induce various epigenetic memories depending on microorganism nature. Constitutively associated transcription factors (TFs) include CCAAT enhancer binding protein beta (C/EBPb), purine rich DNA binding protein (PU.1), early growth response 2 (EGR2) and signal transducer and activator of transcription 1 (STAT1) while hypoxia-inducible factors (HIFs) and interferon regulatory factor 1 (IRF1) may stimulate iron acquisition in pro-inflammatory conditions. Mouse orthologous locus is generally conserved; chromatin patterns typify a de novo myelo-monocytic gene whose expression is tightly controlled by TFs Pu.1, C/ebps and Irf8; Irf3 and nuclear factor NF-kappa-B p 65 subunit (RelA) regulate expression in inflammatory conditions. Functional differences in the determinants identified at these orthologous loci imply that species-specific mechanisms control gene expression.

Nramp1 gene expression in different tissues of Meishan piglets from newborn to weaning

Natural resistance-associated macrophage protein gene 1 (Nramp1) plays an important role in the innate immune response of swine, and is believed to influence disease resistance. In this study, a real-time quantitative polymerase chain reaction technique was used to investigate Nramp1 expression in 12 different tissues in newborn and 7-, 14-, 21-, 28-, and 35-day-old Meishan piglets. Results indicated that Nramp1 was expressed to varying degrees in all sample tissues, although expression differed among growth stages. For example, Nramp1 was highly expressed in the spleen, but minimally expressed in heart, liver, and muscle tissues among the various piglet age classes. Overall, Nramp1 expression increased with age, reaching significant levels in 21- and 28-day-old animals. Nramp1 was expressed in all 12 tissues tested; however, expression in spleen, lung, kidney, and thymus tissues was highest among newborns, which is consistent with this gene's role in innate immunity improvement. Before and after weaning, Nramp1 was highly expressed in digestive (stomach) and intestinal (duodenum, jejunum, and ileum) tissues, further indicating a genetic role in both immune regulation to compensate for weaning stress and enhanced development of intestinal immunity.

Temporal and anatomical host resistance to chronic Salmonella infection is quantitatively dictated by Nramp1 and influenced by host genetic background.

The lysosomal membrane transporter, Nramp1, plays a key role in innate immunity and resistance to infection with intracellular pathogens such as non-typhoidal Salmonella (NTS). NTS-susceptible C57BL/6 (B6) mice, which express the mutant Nramp1D169 allele, are unable to control acute infection with Salmonella enterica serovar Typhimurium following intraperitoneal or oral inoculation. Introducing functional Nramp1G169 into the B6 host background, either by constructing a congenic strain carrying Nramp1G169 from resistant A/J mice (Nramp-Cg) or overexpressing Nramp1G169 from a transgene (Nramp-Tg), conferred equivalent protection against acute Salmonella infection. In contrast, the contributions of Nramp1 for controlling chronic infection are more complex, involving temporal and anatomical differences in Nramp1-dependent host responses. Nramp-Cg, Nramp-Tg and NTS-resistant 129×1/SvJ mice survived oral Salmonella infection equally well for the first 2–3 weeks, providing evidence that Nramp1 contributes to the initial control of NTS bacteremia preceding establishment of chronic Salmonella infection. By day 30, increased host Nramp1 expression (Tg>Cg) provided greater protection as indicated by decreased splenic bacterial colonization (Tg<Cg). However, despite controlling bacterial growth within MLN as effectively as 129×1/SvJ mice, Nramp-Cg and Nramp-Tg mice eventually succumbed to infection. These data indicate: 1) discrete, anatomically localized host resistance is conferred by Nramp1 expression in NTS-susceptible mice, 2) restriction of systemic bacterial growth in the spleens of NTS-susceptible mice is enhanced by Nramp1 expression and dose-dependent, and 3) host genes other than Nramp1 also contribute to the ability of NTS-resistant 129×1/SvJ mice to control bacterial replication during chronic infection.

Removal of the phage-shock protein PspB causes reduction of virulence in Salmonella enterica serovar Typhimurium independently of NRAMP1

The phage-shock protein (Psp) system is believed to manage membrane stress in all Enterobacteriaceae and has recently emerged as being important for virulence in several pathogenic species of this phylum. The core of the Psp system consists of the pspA-D operon and the distantly located pspG gene. In Salmonella enterica serovar Typhimurium (S. Typhimurium), it has recently been reported that PspA is essential for systemic infection of mice, but only in NRAMP1(+) mice, signifying that attenuation is related to coping with divalent cation starvation in the intracellular environment. In the present study, we investigated the contribution of individual psp genes to virulence of S. Typhimurium. Interestingly, deletion of the whole pspA-D set of genes caused attenuation in both NRAMP1(+) and NRAMP1(-) mice, indicating that one or more of the psp genes contribute to virulence independently of NRAMP1 expression in the host. Investigations of single gene mutants showed that knock out of pspB reduced virulence in both types of mice, while deletion of pspA only caused attenuation in NRAMP1(+) mice, and deletion of pspD had a minor effect in NRAMP1(-) mice, while deletions of either pspC or pspG did not affect virulence. Experiments addressed at elucidating the role of PspB in virulence revealed that PspB is dispensable for uptake to and intracellular replication in cultured macrophages and resistance to complement-induced killing. Furthermore, the Psp system of S. Typhimurium was dispensable during pIV-induced secretin stress. In conclusion, our results demonstrate that removal of PspB reduces virulence in S. Typhimurium independently of host NRAMP1 expression, demonstrating that PspB has roles in intra-host survival distinct from the reported contributions of PspA.

Early immune gene expression responses to Salmonella enteritidis infection in indigenous chickens

Salmonella enteritidis (SE) is a common cause of food-borne disease in humans and loss of growth in poultry. An effective method to inhibit salmonellosis is to increase the genetic resistance of poultry to Salmonella through genetic selection programmes that may be performed using phenotypic or genotypic data. A better understanding of the effects of Salmonella infection on the expression of inflammatory and anti-infectious cytokines and antimicrobial molecules is essential for choosing potential markers in selection programmes. The aim of this study was to investigate the expression of NRAMP1, TLR4, IL8 and IFNg genes in the caecum and spleen of Malaysian village chickens and red jungle fowl 48 h after inoculation with SE. Real-time reverse transcription PCR was used to quantify the fold-change in mRNA expression. The results showed that all the genes were highly expressed 48 h post-infection in the caecum of the village chickens. Overall, these results showed that Malaysian indigenous chickens have appropriate innate immune responses to SE infection.

Cell-Type Specific Determinants of NRAMP1 Expression in Professional Phagocytes.

The Natural resistance-associated macrophage protein 1 (Nramp1 or Solute carrier 11 member 1, Slc11a1) transports divalent metals across the membrane of late endosomes and lysosomes in professional phagocytes. Nramp1 represents an ancient eukaryotic cell-autonomous defense whereas the gene duplication that yielded Nramp1 and Nramp2 predated the origin of Sarcopterygians (lobe-finned fishes and tetrapods). SLC11A1 genetic polymorphisms associated with human resistance to tuberculosis consist of potential regulatory variants. Herein, current knowledge of the regulation of SLC11A1 gene expression is reviewed and comprehensive analysis of ENCODE data available for hematopoietic cell-types suggests a hypothesis for the regulation of SLC11A1 expression during myeloid development and phagocyte functional polarization. SLC11A1 is part of a 34.6 kb CTCF-insulated locus scattered with predicted regulatory elements: a 3' enhancer, a large 5' enhancer domain and four elements spread around the transcription start site (TSS), including several C/EBP and PU.1 sites. SLC11A1 locus ends appear mobilized by ETS-related factors early during myelopoiesis; activation of both 5' and 3' enhancers in myelo-monocytic cells correlate with transcription factor binding at the TSS. Characterizing the corresponding cis/trans determinants functionally will establish the mechanisms involved and possibly reveal genetic variation that impacts susceptibility to infectious or immune diseases.

Identification and Functional Characterization of Leishmania donovani Secretory Peroxidase: Delineating Its Role in NRAMP1 Regulation

Leishmania silently evades host immune system and establish in the hostile environment of host macrophage phagolysosomes. For differentiation, growth and division parasite acquires divalent cations especially iron from the host nutritive pool. Natural resistance associated with macrophage protein1 (NRAMP1), a cation transporter that effluxes out divalent cations specifically iron from phagosomal milieu to the cytosol, to create ions deprived status for pathogenic microorganisms. The mechanisms of NRAMP1 regulation are largely unknown in leishmanial infections. In the present study, we identified a secretory Leishmania donovani peroxidase (Prx) that showed peroxidoxin like peroxidase activity and significantly reduced H2O2, O2.− and NO levels in LPS activated macrophages. Further, we also observed down regulated Nramp1 expression and concomitantly declined labile iron pool in activated macrophages treated with identified peroxidase. Prx also decreased levels of TNF-α, IFN-γ and IL-12 in LPS activated macrophages. These observations indicate a bifunctional protective role of secretory Prx; first it reduces redox activation of macrophages, and secondly it allows iron access to Leishmania by down regulating NRAMP1 expression.

Slc11a1 (Nramp1) impairs growth of Salmonella enterica serovar typhimurium in macrophages via stimulation of lipocalin-2 expression

The expression of the cation transporter Nramp1 (Slc11a1) in late phagolysosomes confers resistance to infection with several intracellular pathogens, such as Salmonella enterica, in mice. The antimicrobial actions of Nramp1 are attributable, in part, to modulation of macrophage immune function and cellular iron metabolism--the latter affecting the availability of the essential nutrient iron for intraphagosomal bacteria. Here, we provide novel evidence that Nramp1 functionality increases the expression of the peptide Lcn2, which exerts its antimicrobial activity by scavenging iron-loaded bacterial siderophores and mediating iron efflux from macrophages. With the use of macrophage cell lines expressing functional or nonfunctional Nramp1, we found significantly elevated Lcn2 mRNA and protein levels in Nramp1-expressing cells. These resulted from Nramp1-mediated alterations in the production of ROS, which stimulated NF-κ B activity and subsequently, Lcn2 transcription. We observed that increased Lcn2 levels in primary Nramp1-positive macrophages resulted in a significant suppression of S. enterica serovar typhimurium growth. Stimulation of Lcn2 expression is a novel mechanism by which Nramp1 confers resistance against infection with the intracellular bacterium S. typhimurium.

High Levels of Tumor Necrosis Factor-α Downregulate Antimicrobial Iron Transport Protein, Nramp1, in Chronic Hemodialysis Patients: A Key Factor for Infection Risk

Background/Aims: The susceptibility of patients on maintenance hemodialysis (MHD) to infections is a major cause of mortality and morbidity. Natural resistance-associated macrophage protein 1 (Nramp1) regulates intracellular pathogen proliferation, and its mRNA expression is highest in polymorphonuclear leukocytes (PMNLs). The purpose of this study was to determine the level of Nramp1 in PMNLs from MHD patients and the factors affecting its expression. Methods: Twenty MHD patients and 24 healthy volunteers (controls) were recruited. Relative quantitative PCR was used to measure Nramp1 mRNA, and protein levels were semiquantified by means of real-time PCR and Western blot analysis or immunohistochemistry. The effect of tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6) on Nramp1 expression in PMNLs from controls was also examined. Results: Nramp1 mRNA and protein levels were substantially lower in PMNLs from MHD than control subjects. Serum TNF-α levels were significantly higher in the MHD group and were inversely correlated with Nramp1 mRNA levels. The addition of TNF-α to PMNLs from control subjects decreased mRNA and protein levels of Nramp1. IL-6 did not alter Nramp1 mRNA or protein expression. Conclusion: We found that Nramp1 was downregulated in the PMNLs of MHD patients, which constitute the first defense barrier against bacterial challenges. High levels of TNF-α may be associated with the downregulation of Nramp1. Our findings indicate that the susceptibility to infection observed in MHD patients could be partly due to the impairment of the intracellular handling of iron and the donation of more iron to the bacteria.

Function identification of bovine Nramp1 promoter and intron 1

The Nramp1/Slc11a1 locus encodes a proton-coupled divalent cation transporter, expressed in late endosomes/lysosomes of macrophages, that constitutes a component of the innate immune response to combat intracellular pathogens and it was shown to play an important role in regulating inherent immunity. The previously identified Z-DNA forming polymorphic repeat(GT)n in the promoter region of the human Nramp1 gene does act as a functional polymorphism influencing gene expression. Research has shown that INF-γ, TNF-α, IL-1β and bacteria LPS increase the level of Nramp1 expression. However, the molecular mechanism for Nramp1 gene regulation is unclear. In this research, bovine Nramp1 5′-flanking region (−1748~+769) was cloned and analyzed by bioinformatics. Then to find the core promoter and the cis-acting elements, deletion analysis of promoter was performed using a set of luciferase reporter gene constructs containing successive deletions of the bovine Nramp1 5′-flanking regions. Promoter activity analysis by the dual luciferase reporter assay system showed that the core promoter of Nramp1 was located at +58~−89 bp. Some positive regulatory elements are located at −89~−205 bp and −278~−1495 bp. And the repressor elements were in region −205~−278 bp, intron1 and −1495~−1748 bp. LPS-responsive regions were located at −1495~−1748 bp and −278~−205 bp. The present study provides an initial effort to explore the molecular mechanism of transcriptional activation of the bovine Nramp1 gene and should facilitate further studies to decode the complex regulatory process and for molecular breeding for disease resistance in bovines.