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.The Natural resistance-associated macrophage protein 1  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  Nramp was identified by positional cloning of the dominant gene responsible for the macrophage quantitative trait of natural resistance to infection by unrelated bacterial and protozoan pathogens .2+) and manganese (Mn2+) [Nramp proteins are classified as Solute carriers 11 (Slc11) that mediate chemiosmotic uptake of divalent metals such as ferrous iron Fe+ and mane (Mn2+) . Nramp te (Mn2+) . Accordie (Mn2+) . Hence, Nramp genes such as the amoeba Dictyostelium discoideum, the read alga Cyanidioschyzon merolae and the lower plant (moss) Physcomitrella patens and fungi such as Chytridiomycota, Kickxellomycotina and Mucoromycotina [Saccharomyces cerevisiae) and include the likely source of gene transfer towards prokaryotes. Archetype Nramp are found in multi-cellular organisms and yielded in animals Nramp1 and Nramp2. In the amoeba D. discoideum which grazes on bacteria, both prototype and archetype Nramp contribute to cytoplasmic iron uptake and host defense against bacterial invasion [Bacterial Nramp homologs function as proton-dependent Mn transporters (MntH) but display polyphyletic origins that suggest derived evolution. One group of MntH sequence is restricted to anaerobic micro-organisms; another group possibly emerged later is widespread among Bacteria and includes Archaea, whereas the third group was apparently derived by horizontal gene transfer from a eukaryotic source and is more prevalent among bacteria associated with eukaryotic cells . Few eukmycotina  (DOE JGIinvasion .D. discoideum archetype Nramp (DdNr1) is expressed in intracellular vesicles of the endo-lysosomal pathway. DdNr1 is recruited to phagosomes and macropynosomes where it mediates resistance to invasion by diverse intracellular pathogens such as the Gram positive and negative species Mycobacterium and Legionella, respectively. DdNr1 transport of divalent metals such as ferrous iron out of the phagosome towards the cytoplasm is supported by the electrogenic V-H+ ATPase [D. discoideum archetype Nramp (DdNr1) are thus highly similar to animal Nramp1 [+ ATPase . The intl Nramp1 .D. discoideum prototype Nramp (DdNr2) is expressed in the membrane of the contractile vacuole [DdNr1,2 affects the growth of D. discoideum in conditions of iron depletion and/or overload. Both proteins co-localize with the electrogenic V-H+ ATPase that extrudes protons from the cytoplasm, so that H+ can re-enter as a driving force for metal uptake. Food starvation induces a developmental process in which the amoeba recycles intracellular material to differentiate and produce resistant spores. D. discoideum development is perturbed as a result of deletion of either prototype or archetype Nramp genes [D. discoideum Nramps, and suggest that the ancestral eukaryote gene duplication enabled diversification from nutritive function (prototype) to nutriprive activity (archetype). In fact, archetype Nramp exerts both functions, for phagocytic meal and resistance to environmental conditions or intracellular infection. Such dual role is very similar to Nramp1 roles in recycling iron from ingested erythrocytes and depriving ingested microbes from direct access to Fe and Mn inside the phagosome.In contrast,  vacuole . Deletiomp genes . The dat2+ and Mn2+ [Nramp2/Dmt1 mutations are responsible for microcytic anemia with hepatic overload in humans resulting from Transferrin cycle dependent defect leading to iron accumulation within endosomes [Nramp1 Slc11a1) was characterized as a divalent metal importer expressed specifically in the membrane of late endosomes/lysosomes of professional phagocytes [a1 was chand Mn2+  and mediand Mn2+ ,14. Nramand Mn2+ . Human Nndosomes .Nramp1 and Nramp2 can be traced to the origin of Sarcopterygians. Genome sequencing of the lobe-finned fish Latimeria chalumnae  revealed the coding of Nramp1 and 2 parologs similar to those founds in tetrapods, whereas Actinopterygian (ray-finned) fishes all possess only  Dmt1/Nramp2 homolog(s) , and up to 70% amino acid identity with Nramp2/Dmt1 parologs. In contrast, L. chalumnae Nramp2 protein sequence displays about 80% identity with both tetrapod and ray-finned fish orthologs. Five sites in the predicted TMS2, 3, 6, 8 and 10 show specific variations whose co-occurence distinguishes Nramp1 orthologs from Nramp2/Dmt1 parologs (not shown).The gene duplication that yielded molog(s) . Animal The coelacanth diverged early from the Sarcopterygian lineage  . From a Iron deposition into mature enamel is important for tooth constitution and Fe is the only element detected as deposited by ameloblasts outlining the enamel surface of the teeth . ExistinNramp gene duplication later at the time of emergence of Sarcopterygii would imply highly accelerated sequence evolution for Nramp1.In bony fishes, Fe concentrations in the enameloid were related to the phylogeny of fish and it was proposed that the mechanism of Fe concentration appeared early and disappeared later in the course of fish evolution . RegardiNramp1 and 2 genes in Sarcopterygii. Expression of Nramp2 in the apical membrane of ameloblasts combined to Fpn1 localization in the basolateral membrane would provide a setting that resembles intestinal enterocytes, allowing trans-cellular movement of iron and leading to dental deposition of the metal. It should be instructive to further study the tissue specificity of lobe-finned fish Nramp1 and 2 gene expression as well as and their relative contribution to host defense against infections.It might thus be suggested that ensuring novel house-keeping functions such as iron deposition in dental enamel contributed to maintaining both D. discoideum [The professional phagocyte phenotype of natural resistance to infection by intracellular pathogens which is shared by mouse macrophages  and the scoideum  implies scoideum . AccordiM. tuberculosis (Mtb) is a successful pathogen, responsible for the worldwide human infection tuberculosis (TB), which is latent in more than 2 billion people. Up to 10% may develop TB during their lifetime; each year untreated infections cause the death of over 1.5 million people [n people ,29. Mtb n people ,29,30.IL-12Rβ1, IFN-γR1, IL-12p40, STAT-1, IFN-γR2 or NEMO were traced back to a rare congenital syndrome, Mendelian susceptibility to mycobacterial diseases involving weakly virulent species [IL-12Rβ1) [H1) response that in turn promotes M1 macrophage microbicidal activities; in addition TNFα broadly modulates macrophage activities such as cytokine and chemokine secretions, microbial killing, programmed necrosis or apoptosis. Despite this antimicrobial potential anti-inflammatory cytokines including IL-10 and TGF-β can also be produced by Mtb-infected macrophages, translating an alternative (or M2) state of activation. The resulting downregulation of proinflammatory cytokines and T-cell proliferation and activation leads to a balanced response, which contains bacteria unless external factors perturb that equilibrium and reactivate infectious disease [In general, immunity to mycobacterial species depends on the activation of macrophage microbicidal activities through the IL-12/IL-23–IFN-γ axis. Genetic deficiencies in  species  or to diL-12Rβ1) . Ingesti disease ,33.Strong evidence supports a critical role for genetic factors in susceptibility or resistance to TB but the mechanisms involved remain elusive. Host resistance to TB is a complex trait due to an intricate balance of host-Mtb interactions involving multiple genes and compounding factors . TB resistance has been associated with several gene polymorphisms but with low consistency between replicate studies, while most clinical association results are not yet validated functionally by molecular studies ,35,36.NRAMP1 may be a factor of critical importance for host defense to the development of TB because several NRAMP1/SLC11A1 genetic polymorphisms were consistently associated with TB resistance/susceptibility in many studies, including the disseminated form of pediatric TB [SLC11A1 consistent association with host resistance to developing TB however has not established whether this link regards progression from latent infection to active TB disease or susceptibility to primo-infection [atric TB ,38,39. Snfection ,37,38,40Genetic variants under evolutionary selection often have accumulated to relatively high frequencies, which might represent an equilibrium between benefits procured for TB resistance and high risk of other diseases, such as adverse consequences of microbicidal activities . SignifiSLC11A1  show stronger associations for western populations, and the frequency of the non coding deletion allele 1729 + 55del4 [SLC11A1 resistant alleles by natural selection [SLC11A1 that is predicted to contain regulatory elements  of SLC11A1 exon 3, which was identified as a major risk factor to acquire pediatric TB [SLC11A1 -274C allele on protein function, an assay measuring intracellular recruitment to the phagosome of the macrophage late endosomal/lysosomal marker mannose 6-phosphate receptor (M6PR) was employed [M. bovis BCG, Salmonella enterica Serovar Typhimurium and Leishmania donovani albeit in different ways, depending on the pathogen intracellular strategy [In few instances atric TB . To studemployed . Mouse mstrategy .Salmonella establishes a replicative niche by subverting the normal process of phagolysosomal maturation and excluding for instance, the M6PR from the enclosing vacuole membrane. Assaying M6PR recruitment to the phagosome in human macrophages provided readout of SLC11A1 activity suggesting a link between SLC11A1 -274C allele and decreased innate resistance to pediatric TB [+ monocytes (MNs) and monocyte-derived macrophages (MDMs), might suggest interference with gene regulation  or a given immune context (pro- or anti-inflammatory). In both human histiocytic U-937 cells and the monocytic cell line THP-1 SLC11A1 variant -237C stimulates promoter activation, while hypoxia-inducible factor 1 alpha (HIF-1α) regulates NRAMP1 expression levels by interacting with a microsatellite repeat under evolutionary selection pressure in the promoter region [Hence, several r region ,48,49.SLC11A1 promoter region contains several polymorphisms influencing gene expression. The role of a (GT)n microsatellite repeat consisting of 9 identified allelic variants was characterized in details regarding the two most frequent alleles. These alleles differ by one GT repeat, a variation that affects SLC11A1 promoter activity: allele 3 contains 9 repeats; it is the most frequent allele and drives higher gene expression levels in transient transfection assays using U-937 and THP-1 cells, while allele 2 with 10 repeats has higher Z-DNA-forming propensity but drives lower expression levels in transient transfection assays [The n assays ,48,49,50SLC11A1 allele 3 but not allele 2 bound the transcription factors ATF-3 and c-Jun [SLC11A1 allele 3 transcriptional activation was decreased by co-transfecting ATF-3, an effect that was amplified in cells activated with bacterial lipopolysaccharide (LPS) suggesting that ATF-3 dimer may act as repressor [Z-DNA formation can facilitate transcriptional initiation and activation. This left-handed conformation of the double helix may be formed at TG repeats within promoters upon remodeling of a mononucleosome by the human SWI/SNF complex . Interesnd c-Jun . SLC11A1epressor  , tumour necrosis factor-alpha and interleukin-6 and increasing interleukin-10 production [SLC11A1 genetic polymorphism was correlated with variation in LPS-induced IL-10 secretion [SLC11A1 promoter (GT)n repeat alleles 2 and 3 represent an equilibrium between the survival advantage of TB resistance and increased risk of auto-immune and inflammatory diseases such as type 1 diabetes [Alleles other than allele 3 were significantly associated with an increased risk of TB, independent of disease type, with a meta-analysis mean odd ratio of 1.31 comparing cases to controls  . Impaireoduction . It has oduction , productoduction ,55 and ioduction ,57; it aoduction . Conversecretion . Becauseecretion ,63,64,65diabetes ,66.SLC11A1 promoter (GT)n repeat. HIFs are O2 sensitive transcription factors mediating transcriptional adaptation to hypoxic environments [2, HIFs can translocate to the nucleus and dimerize with their obligate partner Aryl hydrocarbon receptor nuclear translocator (ARNT) a.k.a. HIF-1β , then recruit coactivators such as histone acetyltransferase (HAT) activities . HIF heterodimers activate genes involved in adaptation to hypoxic stress through recognition of and binding to hypoxia-response elements in their promoter. More broadly, HIFs regulation senses oxygen availability, redox status, nutrient availability, as well as inflammatory signals. Hence HIFs integrate metabolic and innate immune responses to infection and inflammation; HIF1α activity predominates in M1 macrophages whereas HIF-2α is preponderant in M2 macrophages [The hypoxia-inducible factor alpha (HIF-1α) represents a strong candidate transcriptional activator binding at ronments . When strophages .HIF-1α gene expression and for HIF-1α protein accumulation in response to hypoxia; also, HIF-1α is activated during in vitro differentiation of MNs into macrophages. Microbial pathogens such as Gram-positive  and Gram-negative  bacteria, as well as protozoan parasites  and viruses induce HIFs in normoxia [HIF-1α is key for M1 microbicidal macrophage functions since HIF-1α basal activity can be stimulated in non hypoxic conditions by a variety of compounds such as growth factors, cytokines, nitric oxide, LPS and a range of infectious microorganisms ,69. NF-Κnormoxia ,69.SLC11A1 proximal promoter (GT)n repeat variation regulates allele expression. This microsatellite contains two predicted hypoxia responsive elements and demonstrates Z-DNA-forming ability both in vitro and in vivo. Transient co-transfections in Baby hamster kidney cells revealed that under normoxic conditions HIF-1α induced up to 4-fold increase in luciferase expression while HIF-2α could not transactivate NRAMP1 promoter, and SLC11A1 expression levels after transfecting Chinese hamster ovary cells depended on the presence of HIF-1α. In murine macrophages co-transfected with HIF-1α and SLC11A1 proximal promoter transcriptional activity was upregulated in response to hypoxia mimetics and HIF stabilizers . The microsatellite allele 3 binds directly the transcriptional regulator HIF-1α/ARNT heterodimers in vitro, and in vivo when THP-1 macrophages are activated by pathogen or proinflammatory signals [ signals .SLC11A1 promoter cis-acting regulatory variation as well as synonymous coding polymorphism can possibly contribute to heritable differences in gene/protein activity that may account for increased resistance/susceptibility to TB. In this regard, appraisal of all the regions involved in the transcriptional regulation of SLC11A1 will facilitate localizing functional SNPs that may influence with gene expression.Current genetic data thus suggest that SLC11A1 (NRAMP1) is tissue-specific expression, limited to the myelo-monocytic lineage [SLC11A1 expression is thus likely regulated by factors involved in myeloid cell differentiation and/or which may affect phagocyte polarization toward pro- or anti-inflammatory phenotypes [A hallmark of  lineage . The hum lineage ,71,72. H lineage ,74 or in lineage . Based oenotypes .The myeloid lineage is part of the haematopoietic system which comprises blood cells and is organized hierarchically. Under the current paradigm of hematopoietic differentiation blood cell types arise from hematopoietic stem cells (HSC) which can self-renew or evolve through multiple progenitor and intermediate maturation states into 12 terminally differentiated cell types ,77. PhenmRNA profiling of intermediate and terminally differentiated cell types revealed that genes are tightly coexpressed in modules which are restricted to specific lineages or common to multiple hematopoietic lineages and interconnected. Five dominant phenotypes: HSPCs, differentiated erythroid cells, granulocytes/MNs, B cells and T cells are distinguished by a set of differentially expressed genes specific to each lineage as compared to the others . AnalyseSLC11A1 high levels of expression in blood neutrophils and MNs prompted testing the promyelocytic cell line HL-60 as a model to study SLC11A1 regulation during either monocytic or granulocytic differentiation pathways using various pharmacological agents [SLC11A1 transcript accumulation was detected co-induced with selected marker genes after 4–6 days differentiation into macrophage- , monocyte- OH2 VitD3, VitD, CD14) or granulocyte-  like cells, suggesting that SLC11A1 expression occurred late in the myelo-monocytic differentiation program. Accordingly, SLC11A1/NRAMP1 was detected in tertiary granules of neutrophils [all-trans retinoic acid (ATRA), classically used for leukemia differentiation therapy [SLC11A1 expression is induced along either mono- or granulocytic pathways, and protein detection in the membrane of phagosomes of both types of phagocytes supports that SLC11A1 constitutes a functional marker of professional phagocytes [SLC11A1 expression, presumably by activating particular (combination of) transcription factors.l agents . SLC11A1trophils . However therapy . The datagocytes ,72. The 2 VitD3, VitD) binds the vitamin D receptor (VDR) and the VitD-VDR complex assembles with the retinoic X receptor  [Vitamin D active metabolite OH-cis RA)  or with -cis RA) ,84. Dime-cis RA) .SLC11A1 expression despite inducing granulocytic maturation (ATRA) [cis RA) [NRAMP1 expression was more efficiently up-regulated specifically in the presence of VitD genomic vs. non-genomic agonists [SLC11A1 expression was found slow and moderate compared to the monocyte marker CD14, implying perhaps an indirect process mediated by a VitD-induced factor that would bind to and regulate SLC11A1 promoter.Retinoids do not induce n (ATRA)  or repre[cis RA) . Since Vagonists , VDR-depagonists ,87,88. SSLC11A1 by VitD may have physiological implications since this secosteroid hormone stimulates, through VDR binding, myelopoiesis and the maturation of MNs towards macrophages with an M2—or anti-inflammatory—phenotype [The regulation of henotype ,90. VitDhenotype ,94,95,96henotype ,94,97,98henotype .Local productions of VitD by epithelial and immune cells as well as adipocytes exert autocrine or paracrine immunomodulating effects ,96,99. HH1 cell-secreted IFN-γ induces VitD antimicrobial pathway , which is similar to the response triggered by TLR2/1 ligands . Both rely on monocyte secretion of IL-15 albeit through different pathways (STAT1 or MyD88). Monocytes stimulation by IFN-γ depends on the presence of sufficient VitD serum precursor as IFN-γ stimulates CYP27B1 activity and treatment with IFN-γ is inhibited by a VDR antagonist. In addition, IFN-γ-induced autophagy and phagosome maturation are VDR-dependent. A concentration superior to 45 nM of 25OH VitD3 is strictly required to observe IFN-γ—and IL-15-dependent monocyte antimicrobial responses . VitD appears thus instrumental for acquired immunity to activate macrophage antimicrobial responses that overcome intracellular pathogens evasion strategy [This paracrine antimicrobial role of VitD is required for IFN-γ-mediated microbicidal activity of human macrophages. Tstrategy .SLC11A1 mRNA in response to IFN-γ. However, once isolated from its native chromatin environment SLC11A1 proximal promoter became directly responsive to IFN-γ treatment for two days, even though the cytokine induces little monocytic differentiation [SLC11A1 may be part of the antimicrobial arsenal that is primed by VitD (or genomic agonists) and VDR, whose activity increases SLC11A1 proximal promoter accessibility and responsiveness to transcriptional stimulation by antimicrobial factors such as IFN-γ. SLC11A1 sequence 647 bp upstream of the ATG comprises a basal promoter element, starting 263 bp 5' of the ATG, which alone drives maximal transcriptional activity in non myeloid background  and independent of VDR agonist. More distal upstream elements are required for maximal promoter expression level in promyelocytic HL-60 cells and to obtain further VDR-dependent transcriptional upregulation [In HL-60 promyelocytic cells, inducing differentiation with VitD was required to obtain upregulation of the accumulation of the endogenous ntiation . Thus SLgulation .cis-regulatory elements by “opening” inaccessible chromatin domains, as their binding initiate nucleosome remodeling and prime targets genes for expression in cell-type-specific manner [SLC11A1 lacks conventional TATA or CAAT box and other signals generally important for transcription activation [SLC11A1 candidate promoter region 647 bp upstream of the ATG and DNAse I footprinting analysis allowed to test the hypothesis that transcription factors gain access and bind to this region during myelo-monocytic differentiation.During differentiation various epigenetic marks decorate the genome to demarcate domains of activity that are affected in a hierarchical manner. For instance, HSC pluripotency genes get progressively turned off with the onset of differentiation, by recruiting histone deacetylases and affecting histone methylation pattern to induce formation of heterochromatin . Conversc manner . SLC11A1tivation . ProgresSLC11A1 major transcription start site (TSS) was mapped by 5' RACE PCR and S1 mapping in monocytic THP-1 cells and HL-60 cells differentiated into MNs, respectively [cis-acting sites required for myelo-monocytic expression and the binding factors they recruit were identified. Double stranded DNA probes corresponding to footprints protected in vitro from DNAse I digestion after incubation with nuclear extracts from differentiated HL-60 cells were characterized by electromobility shift and supershift assays. The candidate cis elements were tested in vitro by site-directed mutagenesis and the effect of linker-mutations was verified by reporter assays after transfection or co-transfection of promoter constructs with selected transcription factors. Interaction of the candidate factor with the promoter area containing the site defined was verified by chromatin immuno-precipitation (ChIP) assay. The results showed that SLC11A1 major TSS is adjacent to a 5' C/EBP binding site, which is required for transcriptional activation during monocytic and granulocytic differentiation, and within an area bound by C/EBPα in promyelocytic cells and by C/EBPβ beginning 24 h after induction of differentiation with VitD. Site-directed mutagenesis of this C/EBP binding site also abrogated transcriptional activity of the promoter in non myeloid background implying it is required for recruiting the basal transcription complex. A binding site for the Specificity protein 1 (Sp1), which transactivates SLC11A1 promoter in vivo, was delineated in the more upstream region of the proximal promoter that is required for myeloid expression [ectively ,109. TwoSeveral members of the C/EBP family of bZIP transcription factors have important roles in myeloid development, and in macrophage in particular, as C/EBPα , C/EBPβ and C/EBPδ represent three of the 18 regulators most frequently represented in the 14 modules enriched for macrophage-related gene signatures . The indSLC11A1 expression in professional phagocytes is controlled by C/EBPs.C/EBPβ (a.k.a. NF-IL6) is a key regulator of monocytic cells development . It servSLC11A1/NRAMP1 gene expression was detected not only in pro-inflammatory (M1) but also anti-inflammatory (M2) macrophages [NRAMP1 expression may relate to steady state activities of tissue macrophages and maintenance of tissue integrity.Regarding macrophages, rophages ,115. Morrophages  and the rophages . C/EBPβ rophages ,90. The rophages  is anothrophages . At homerophages ,118,119.SLC11A1 transcription may support this view because Sp1 has been widely associated with basal levels of constitutive expression, including at various myeloid promoters. Despite being widely expressed the transcription factor Sp1 can regulate the expression of tissue-specific genes through combinatorial effects with a limited number of key transcription factors [C/EBPδ , the complement and pattern recognition receptors CD11b and CD14, respectively, lactoferrin and the Myeloid Elf-1 like Factor (MEF) which codes for an ETS protein that activates the promoters of myeloid effectors and stimulators such as the granulocyte macrophage colony-stimulating factor, interleukin-3, lysozyme, human beta defensin-2 and perforin [SLC11A1 proximal promoter upstream region which is required to confer myeloid specific expression and VDR-dependent upregulation. Mutating one binding site for Sp1 had less impact than abrogating SLC11A1 TSS C/EBPα/β site [SLC11A1 myeloid-specific expression through combinatorial interactions including transcription factors key for myelomocytic differentiation such as C/EBPβ.The contribution of the Specificity protein 1 (Sp1) to regulate  factors . Hence, perforin ,125,126.perforin ,124,126 α/β site ,109. Hencis element key for SLC11A1 expression along macrophage-like differentiation induced with PMA was recently characterized. PMA induces SLC11A1 transcriptional activation in HL-60 cells and the mRNA is stabilized by the interaction of a 3' AU-rich element (ARE) with a ubiquitously expressed RNA-binding protein, Hu antigen R (HuR). PMA-induced migration of HuR from the nucleus to the cytoplasm paralleled increased binding of HuR to SLC11A1 ARE and the accumulation of transcript. Overexpression of HuR in HL-60 cells stabilized SLC11A1 mRNA levels induced after 3 days PMA treatment, whereas siRNA HuR knockdown limited both SLC11A1 transcript and protein accumulation [HuR-null macrophages show exacerbations in the biosynthesis of inflammatory cytokines and chemokines; HuR is essential for balancing proinflammatory response and homeostatic regulation to control the extent of the inflammatory response [Another mulation . The RNAresponse . Given tSLC11A1 transcriptional regulation during macrophage-like differentiation of HL-60 cells pointed at a PMA-responsive element, which was located by nested deletions of a reporter construct including the upstream myeloid-specific region. The PMA responsive element is adjacent in 5' to the (GT)n repeat sequence that is converted into Z-DNA conformation and which contains two binding sites for the HIF-1α/ARNT heterodimer [Progress in understanding molecular mechanisms of erodimer ,129  is a member of the ATF/cyclic AMP responsive element binding family (CREB) family of transcription factors. ATF3 is expressed in a number of splice variants and interacts with multiple partners to form dimeric complexes, acting either as repressor or activator of various genes. For instance in Toll-like receptor (TLR)-activated macrophages, ATF3 recruits the histone deacetylase 1 (HDAC1) to the promoters of genes encoding cytokines or involved in metabolic and inflammatory responses, exerting negative regulation by epigenetic modification . ATF3 ised death . Howevered death . In addipromoter .SLC11A1 promoter stimulates transcriptional activation [Xenopus oocyte nucleus enables to reactivate heterologous embryonic genes, indicating that nuclear actin allows transcriptional activation by de-repressing silenced genes and this regulation is an evolutionary conserved feature [SLC11A1 promoter, enhancing the ATPase activity of the SWI/SNF chromatin remodeling complex to create a Z-DNA structure that may facilitate interaction with HIF-1α/ARNT heterodimers and the activation of SLC11A1 transcription in the days following induction of differentiation with PMA.The recruitment of β-actin to tivation . The bastivation . It has tivation . Cell di feature . AccordiSLC11A1 transcriptional activation during VitD and PMA differentiation followed similar time frames. Since β-actin translocates from the cytoplasm to the nucleus after two days differentiation induced with PMA and, because C/EBPs can also recruit the SWI/SNF chromatin remodeling complex, it would be interesting to determine if VitD-induced SLC11A1 transcription depends also on β-actin stimulation. Alternatively the mode of transcriptional activation could vary depending on the developmental program since C/EBPα was shown to bind SLC11A1 TSS in undifferentiated promyelocytic cells. In contrast, NRAMP1 Z-DNA interaction with ATF-3 and HIF-1α/ARNT were evidenced in cells more advanced along the monocytic differentiation pathway, and in response to typical M1 macrophage activating stimuli such as LPS and IFN-γ [galectin-1, also regulated by HIF-1α, which interacts with and enhances the transcriptional activity of C/EBPα [nd IFN-γ . Yet botnd IFN-γ . In monof C/EBPα .SLC11A1 transcription early in the myelo-monocytic development program may require as well recruitment of the co-activator Mediator multiprotein complex, which acts as molecular bridge between promoter-bound activators and the core transcriptional machinery. Both C⁄EBPβ and Sp1 can bind elements of this complex which may serve to integrate signaling toward the transcriptional machinery during cell differentiation. Another possibility would consist in recruiting coactivator complexes with HAT activities such as p300; histone acetylation may either provide binding surfaces for other activator proteins or facilitate chromatin decondensation to increase accessibility to the transcription machinery. However it might be equally important to consider as well the possible contribution of distal elements which may provide critical signals to recruit the basal transcription machinery [Induction of achinery ,141,142.trans-factors leads to a dynamic primed state and partial chromatin remodeling, which then evolves from resting to induced state [HSC differentiation results from a stepwise process that produces cells demonstrating various degrees of lineage potential until reaching a stage of committed progenitor, which yields only one lineage through a multistage process producing mature cells. Because most eukaryotic DNA is packaged in closed, tightly packed chromatin conformation (heterochromatin), chromatin structure has an important role at the local and chromosomal levels in controlling hematopoieteic gene expression by regulating accessibility to the transcription machinery . The cored state ,144. Reged state ,146 sugged state ,147,148.SLC11A1 expression or support results previously obtained following hypothesis-driven approaches.Using the University of California Santa Cruz (UCSC) Genome Browser  to visuaSLC11A1/NRAMP1 localizes at 2q35 in a densely populated chromosomal region between the gene encoding the unknown Orf C2ORF62, upstream, and CTDSP1, coding for the CTD  small phosphatase 1, adjacent to SLC11A1 3' end  are present within SLC11A1, between exons 7–8 and 10–11, while a few CpG dinucleotides were found mainly clustered in the basal proximal promoter region around the TSS [1 3' end A. The th the TSS .SLC11A1 TSS established by independent approaches maps 148 bp 5' of the translational initiation codon. The proximal promoter region of SLC11A1 and those of flanking genes display sequence conservation among vertebrates, together with exons and 3' UTR (SLC11A1 and CTDSP1); some areas in SLC11A1 putative distal promoter also show sequence conservation suggesting that some cis regulatory elements might have been preserved across species.A single predicted transcription start site (SwitchGear Genomics) locates 41 bp downstream the major TSS mapped experimentally ,109. SLCSLC11A1 locus displays several areas that correspond to hypersensitive sites to DNAse I digestion in intact nuclei among 148 cell types, which were identified by direct sequencing of the ends of DNAse I “double-hit” fragments  and not in hepatocyte, lymphocyte or fibroblast cells for instance . ELF-1 controls the expression of multiple essential haematopoietic regulators; its downregulation is necessary for erythrocyte differentiation [SLC11A1 candidate functional polymorphism D543N , which are predicted on similar grounds (DNA sequence fragments hypersentive to DNAse I matching those pulled by ChIP-Seq assay): both candidate sites are covered by a 410 bp genomic footprint; the DNA fragments pulled down by ChIP-Seq share the 5' boundary but STAT3-specific segments extend 150 bp further in 3'. This upstream C/EBP site is equally associated with C/EBPβ and PU.1 both in CD14and MDMs  supportiSLC11A1 expression is reduced in these cells, consistent with observation of limited histone modifications  [STAT3 and C/EBPβ have complementary roles supporting host defense against infection as both are IL-6-regulated transcription factors . C/EBPβ o C/EBPβ , can fun (CREBP) .MAFK is frequently a target of chromosomal translocations associated with acute myeloid leukemia ). It encodes a short polypeptide that, besides acting as a dominant negative protein antagonizing large MAF proteins, can also regulate the transcription of heme oxygenase (HO-1) [SLC11A1 myelo-monocytic expression.The long isoform MAFB induces monocytic differentiation  and preve (HO-1) . Inducede (HO-1) , and thee (HO-1)  allows Me (HO-1) ,171. Alse (HO-1)  implying+ MNs and MDMs, albeit with moderate intensity, and EGR-2 co-binding was reported in MDMs as well  is a ubiquitous c-Myc-related regulatory factor required for M6PR promoter activity . The earcis-acting determinant  and ELF-1 (downstream) may contribute to regulate gene transcription during myeloid differentiation leading to mature myelo-monocytic cells. This proposition is supported by observing that both these elements are part of areas decorated by histone modification marks which correlate with cell-type specific regulatory elements  ~18 and 10 kb upstream SLC11A1 TSS. Indeed, Footprint #4A likely represents a PU.1 binding site in terminally differentiated monocytic cells  was detected in CD14+ MNs, and to lesser extent in HL-60 cells  may contribute to developmental control of SLC11A1 expression, similarly to predicted interactions with PU.1 and ELF-1 .Two other elements potentially important for G2 cells D. The seG2 cells A ,B may SLC11A1 locus of long range myeloid-specific cis-acting determinants additional developmental stages of myelopoiesis were examined using available ENCODE datasets. CD34+ stem cells mobilized with G-CSF are routinely prepared for repopulation of mature myeloid cells after chemotherapy for instance or for hematopoietic transplantation [+ cells present a phenotype close to CMP  cells derived from a patient with Down syndrome. CMK cells differ from the chronic myeloid leukemia (CML) in myeloid blast crisis K-562, as the former exhibits more megakaryocytic properties and the latter erythroid markers . Here agting MEP A,B.SLC11A1 locus spans 34.6 kb and it is activated early in myeloid development. Two candidate elements, both distal from the TSS and located at the ends of SLC11A1 locus, nested within CTCF boundaries, are predicted to bind ETS-related factors such as PU.1 and ELF-1 as early as the CMP stage. The whole locus appears extensively sensitive to DNAse I digestion along the myelo-monocytic pathway, with increasing intensity as maturation progresses. The data support prominent roles for C/EBPβ and PU.1 in the developmental control of SLC11A1 expression in mature phagocytes. Notably three C/EBPα/β sites, including the major TSS and two predicted upstream binding sites appear strongly mobilized to activate transcription in CD14+ MNs and MDMs, while another upstream site seems possibly involved earlier in promyelocytic development (NB4 cells). In contrast, most of the locus internal DNAse I hypersensitive areas disappear along the megakaryo-erythrocytic pathway implying suppression of SLC11A1 expression in this non-phagocytic myeloid lineage.In summary, combined examinations of DNAse I footprinting and ChIP-Sequencing converge to reveal different types of protein-DNA interactions. Depending on cell type(s) in which they were found these DNA-protein associations may occur at different stages of, or be maintained through, the myelo-monocytic differentiation program. Data indicate that Alterations of chromatin structure, such as changes in nucleosome positioning and histone-modification patterns contribute to regulate gene transcription . Using hSLC11A1 transcription may be appreciated by comparing the Chromatin state segmentation results  between fibroblastic (NHLF), hepatocytic (HepG2), B lymphocytic (GM12878) and megakaryotic cells (data not shown).Hence myeloid specificity of SLC11A1 in 3', CTDSP1, is the most active and seems expressed in all cell types considered with by decreasing order: B lymphocytes, fibroblasts, megakaryocytes and hepatocytes. Lymphocytes and fibroblasts show strong enhancer activity and highly active transcription. In contrast, SLC11A1 is inactive in these cell types, either actively repressed by Polycomb proteins in fibroblasts or in the form of genetically inactive and tightly coiled heterochromatin in B lymphocytes (Broad ChromHMM). Though HepG2 chromatin shows weak/poised enhancer activity in the 3' part of SLC11A1, the 5' promoter is rather inactive. The myeloid K-562 nuclear context suggests weak level of gene transcription in the absence of enhancer or promoter activity, and not confirmed in transcriptomic analysis  that are delimited by two strong CTCF binding sites. Among other cell types, the presence of dual methylation marks of transcriptional activation/inactivation in HepG2 cells  may explain lower levels of CTDSP1 transcript  but no evidence of gene transcription such as VIL for instance , which were detected in promyelocytic bi-potential precursors  as well as the distribution of transcription factors potentially interacting with SLC11A1 sequence determinants.To study further SLC11A1 predicted 3' regulatory element, independent data confirm that it is also marked in K-562 megakaryocytic cells  and in NB4 promyelocytic cells (H3K4me3), as well as protein interactions revealed by ChIP-Seq analyses at relevant DNA sites and involving RNA Pol II, c-Myc and Max H, togethSLC11A1 may also increase phagocyte cytoplasmic Fe content it would seem plausible that c-Myc-Max functional complexes have a positive impact on SLC11A1 expression, albeit early in myeloid differentiation. Indeed, significant interactions were detected between c-Myc-Max complexes, RNA Pol II and SLC11A1 locus in promyelocytic NB4 cells  and ferrochelatase (Fech) and increases iron uptake and heme biosynthesis as a result of more c-Myc-Max functional complexes interacting with TfR1 and Fech promoter elements . BecauseB4 cells G. In HL-B4 cells F. These SLC11A1 3' distal elements was found in the chromatin of CD14+ MNs and MDMs  or not (B lymphocytes), and to a lesser extent in lung fibroblasts, but absent from HepG2 cells  which matches DNA fragments pulled by ChIP-Seq that targeted a variety of transcription factors, including RNA Pol II, c-MYC-MAX, EGR-1, ETS-1 and ELF-1. These factors are similar to those potentially binding at the 5' boundary of this large enhancer element, as well as at SLC11A1 proximal promoter cover about half of the coding gene. Starting at the TSS this area extends to a common DNAse I footprint which gave a strong signal in MNs that corresponds in ChIP-Seq analysis of HepG2 cells to DNA associations with transcription factors such as SP1, ELF-1, p300, FOXA1, NR2A2  [SLC11A1 TSS  ,156 allo11A1 TSS B, which 11A1 TSS . In cont  and the other (65) is more common . The presence at these two sites of c-Myc-Max complexes in NB4 and not K-562 cells may thus be interpreted as myelomonocytic-specific recruitment of a transcription factor, which is one of the first steps of enhancer nucleosome remodeling ,190. ThiSLC11A1 transcription: Chromatin opening of SLC11A1/NRAMP1 gene is proposed to start at the locus boundaries. The 3' regulatory element, which is adjacent to a CTCF-specific element that insulates from nearby CTDSP1 gene, may bind “pioneer” transcription factors at an early myeloid stage before divergence of the megakaryo-erythrocytic and myelo-monocytic lineages (CMP stage). The co-existence of DNAse I footprints matching site-specific transcription factor interactions in an area of specific histone methylation and acetylation marks, together predict some activity of this element in megakaryocytic cells. Site-specific associations with transcription factors  imply that this 3' regulatory element is also active in NB4, and hence not sufficient to trigger SLC11A1 transcription in committed myeloid progenitors. Histone modification profile in CD14+ MNs predicts cell-type specific activity of SLC11A1 3' regulatory element, supported by site-specific associations reported with C/EBPβ and EGR-2 [It is thus possible to propose a hypothetical progression of events leading to the activation of nd EGR-2 . It is pSLC11A1 5' enhancer and its 5' boundary are also mobilized early during myeloid differentiation. However, it is only along the myelo-monocytic lineage that SLC11A1 5' enhancer further recruits transcription factors and becomes activated, as seen in DNAse I footprints and histone modification marks from NB4 and HL-60 promyelocytic cells and in CD14+ MNs. Based on site-specific associations with several transcription factors  it is suggested that this upstream enhancer is targeted by multiple sequence-specific transcription factors and becomes fully activated in CD14+ MNs and MDMs, and at least partly in promyelocytic HL-60 cells which demonstrate bound RNA Pol II at SLC11A1 TSS. Consequently, defective activation of SLC11A1 5' myelo-monocytic enhancer (lack of RNA Pol II and/or PU.1 and C/EBPβ association with select sites) may contribute to explain the absence of transcriptional activation at SLC11A1 promoter in NB4 and K-562 cells, which represent progenitors committed towards the myelo-monocytic or megakaryo-erythrocytic pathway, respectively.Similarly, the core of A) and four others distributed over SLC11A1 promoter and first half of the gene body  are less intense in NB4 cells compared to HL-60 and CD14+ MNs. The locations of these footprints correspond in CD14+ MNs to areas where H3 histones are modified by acetylation and exhibit K4me1,2 marks typical of cell-type specific enhancer elements [+ MNs and MDMs. Hence defective activation of myelomonocytic-specific upstream enhancer elements such as Footprint #4A  mediating both functions. All Sarcopterygii including land vertebrates deposit Fe in dental enamel, a process aided by Nramp2/Dmt1 and Fpn1 in mouse ameloblasts. Due to the reaction of Fenton SLC11 activities may impact the production of reactive oxygen species so that their expression also has to be co-ordinately regulated with metal storage and/or export functions as well as redox sensitive, cyto-protective transcriptional responses.SLC11A1/NRAMP1 tissue specific expression is controlled primarily at the transcriptional level and specific sequence determinants in the promoter region have been defined functionally using the myelo-monocytic model cell line HL-60. Monocytic differentiation induced with VitD yields CD14+ cells with functional properties reflecting steady-state anti-inflammatory conditions; they express SLC11A1 under the control of C/EBPβ which binds the major TSS. It is likely that PU.1 is also required to activate SLC11A1 transcription. Macrophage-like differentiation induced with PMA, or activation of MN-like cells with bacterial LPS both stimulate pro-inflammatory functions and SLC11A1 expression; binding of AP-1-like ATF3/JunB dimers allows to recruit the SWI/SNF chromatin remodeling complex that facilitates binding of HIF-1α which upregulates SLC11A1 transcription.cis-elements and trans-acting factors involved at different steps controlling the developmental regulation of gene expression.Genome-wide analyses of chromatin status and transcription factor binding in various tissues have revealed that 80% of the genome exert functional role ,152,178.SLC11A1 allowed to confirm the major TSS indicated by the presence of RNA Pol II in HL-60 myelo-monocytic cells, support the prominent role of C/EBPβ and indicate an important role for PU.1, not only at the TSS but also upstream enhancer elements. The data reveal two distal regulatory elements located close to SLC11A1 locus boundaries and apparently mobilized early during the myeloid developmental program, and allow to predict several regulatory sites within the large 5' enhancer or scattered along the body of SLC11A1 gene. The regulation of SLC11A1 expression is thus significantly more complex than initially anticipated.Examining these and other data regarding SLC11A1 locus is inactive in HSCs but displays activation marks at the CMP stage. Whereas SLC11A1 locus activation recedes in the megakaryo-erythrocytic pathway, the gene acquires along the myelo-monocytic lineage histone modification marks and displays site-specific associations with transcription factors, including RNA Pol II, which together indicate tissue-specific activation of transcriptional activity.Data from cell types representing discrete steps in the myelopoietic pathway also suggest a plausible sequence of events controlling gene expression. SLC11A1 locus between HL-60 and NB4 cells or K-562 and CMK cells, and which in turn may inform on (epi)genetic processes relating to leukemogenesis.Studying leukemia cells representing committed myeloid progenitors with bi-potential differentiation capacity not only enables longitudinal study of the control of gene expression but to also address differences in locus activation, which may result from genetic and/or epigenetic variations such as those observed at SLC11A1 is not prone to transcriptional activation. Epigenetic differences between HL-60 and NB4 cells alter their response to VitD, which induces differentiation by genomic (VDR) or non-genomic mechanisms, respectively, [SLC11A1 locus can thus help evidence molecular aberrations that occur in myelo-monocytic leukemia.Current data confirm that HL-60 cells provide a model representative of human myelo-monocytic differentiation. However, in the other promyelocytic cell line NB4, ctively, ,187,193.SLC11A1 lies within a 34.6 kb insulated locus that comprises an abundance of predicted cis-elements which may exert positive or negative effects on the activity of SLC11A1 proximal promoter and gene expression, not only during myeloid development but also due to phenotypic plasticity of mature cells reaching blood and/or tissues and depending on the immune context, being steady-state or inflammatory conditions. Chromatin remodeling is key to macrophage polarization as the Jumonji-D (JmjD) domain Jmjd3 protein catalyzing H3K27 demethylation to revert gene silencing is essential for M2 macrophage polarization, whereas M1 activation leads to H3K4 trimethylation on cytokine gene promoters, implying active gene transcription [cription .SLC11A1 basal expression in VitD polarized/CD14+ (M2) cells depends on C/EBPβ and correlates with H3K4me3 modification in the absence of H3K27me3 mark. Experimental validation of the developmental control of SLC11A1 expression will provide a useful framework to further analyze how macrophage polarization and response to infectious and/or inflammatory stimuli modulate SLC11A1 transcription. In addition, SLC11A1/NRAMP1 function represents significant pro-inflammatory potential as the protein catalyzes cytoplasmic accumulation of labile iron. To avoid tissue damage such potential pro-inflammatory activity must be tightly regulated in coordination with immune status as well as cytoprotective responses.+ symport [NRAMP1 transcription induced by bacterial LPS or the pharmacological agent PMA thus may contribute to M1 macrophage pro-inflammatory activity.Hence M2 macrophages geared to recycle apoptotic cells and iron, fuel mitochondrial biogenesis and oxidative metabolism but limit the production of reactive oxygen species (ROS). ROS production resulting from mitochondrial respiration is controlled by modulating the expression of uncoupling proteins, e.g., UCP2 , which p symport . Continu symport ,198. In  symport . Up-reguSLC11A1 locus activity in myelo-monocytic cells is expected to further gain in precision and comprehensiveness thanks to continuous large scale efforts aiming to depict all the functional elements that may affect gene expression. Data analyses combining different myeloid cell types provide a road map for future targeted studies aimed at elucidating the mechanisms that govern SLC11A1 transcriptional activation and its regulation by demonstrating the relevant steps experimentally.Understanding the regulation of SLC11A1 SNPs most consistently linked to resistance/susceptibility to TB provides another perspective on modulations of SLC11A1 expression. The proximal promoter comprises a polymorphic (TG)n repeat that contains two sites for the transcription factor HIF-1α which typifies M1, pro-inflammatory macrophages. Activation with LPS, or macrophage-like differentiation of promyelocytes, induce binding of an AP-1 related transcription factor complex (ATF3/JunB) immediately upstream of the (TG)n repeat. The ATF3/JunB complex recruits components of the SWI/SNF chromatin remodeling complex which actively convert the (TG)n repeat conformation into Z-DNA that allows binding of the transcriptional activator HIF-1α. Alleles different from allele 3, which are less efficient at recruiting HIF-1α have been associated with susceptibility to TB.Functional validation of SLC11A1 predicted 3' distal regulatory element also comprises polymorphisms that were linked to TB resistance/susceptibility such as the mutation D543N. The corresponding amino acid replacement is located in a region of the protein lacking sequence conservation and it is not established whether it affects protein activity. But SLC11A1 coding exon (XV) spans a DNA segment hypersensitive to DNAse I digestion in myelo-monocytic cells which was functionally associated with the transcription factors ELF-1, EGR-2 and C/EBPβ. It seems thus possible that SLC11A1/NRAMP1 mutation D543N affects gene expression rather than protein activity. This suggestion is supported by occurrence of a nearby 3' UTR polymorphism that was linked to increased TB resistance in urbanized populations.SLC11A1 gene regulation during myeloid development and/or activation of mature cells is ultimately desirable. These SNPs may perturb transcription factor binding and activity or impede allelic nucleosome marking and remodeling and thus impact gene expression [SLC11A1 locus and pathogenesis of infectious and/or immune diseases.Identifying all the SNPs that are located in areas of open chromatin such DNAse I hypersensitive sites which may influence pression . The ide


Period (Per) genes and the PAR-bZip clock-controlled gene D-site albumin promoter-binding protein (Dbp). These effects are due to cytokine-induced interference of E-box mediated transcription of clock genes. In the present study we have assessed the two E-box binding transcriptional regulators Twist1 and Twist2 for their role in cytokine induced inhibition of clock genes.Activation of the immune system affects the circadian clock. Tumor necrosis factor (TNF) and Interleukin (IL)-1β inhibit the expression of clock genes including Per1, Per2, Per3 and of Dbp was assessed in NIH-3T3 mouse fibroblasts and the mouse hippocampal neuronal cell line HT22. Cells were treated for 4h with TNF and IL-1β. The functional role of Twist1 and Twist2 was assessed by siRNAs against the Twist genes and by overexpression of TWIST proteins. In luciferase (luc) assays NIH-3T3 cells were transfected with reporter gene constructs, which contain a 3xPer1 E-box or a Dbp E-box. Quantitative chromatin immunoprecipitation (ChIP) was performed using antibodies to TWIST1 and CLOCK, and the E-box consensus sequences of Dbp (CATGTG) and Per1 E-box (CACGTG).The expression of the clock genes Twist1 protects NIH-3T3 cells and HT22 cells from down-regulation of Period and Dbp by TNF and IL-1β. Overexpression of Twist1, but not of Twist2, mimics the effect of the cytokines. TNF down-regulates the activation of Per1-3xE-box-luc, the effect being prevented by siRNA against Twist1. Overexpression of Twist1, but not of Twist2, inhibits Per1-3xE-box-luc or Dbp-E-Box-luc activity. ChIP experiments show TWIST1 induction by TNF to compete with CLOCK binding to the E-box of Period genes and Dbp.We report here that siRNA against Twist1 plays a pivotal role in the TNF mediated suppression of E-box dependent transactivation of Period genes and Dbp. Thereby Twist1 may provide a link between the immune system and the circadian timing system. Period (Per), Cryptochrome (Cry), and of the members of the PAR-bZip family of transcription factor genes including the D-site albumin promoter-binding protein (Dbp) [Rev-Erb-alpha, which in turn represses the transcription of Bmal1. These pathways drive the 24-h rhythms in physiology.Clock genes mediate circadian rhythmicity and thereby control behavior, sleep-wake cycles, metabolism and inflammation –3. Synchin (Dbp) , 5. PER Per1, Per2, Per3, Cry1 and Cry2 and of the PAR-bZip clock- controlled genes Dbp, tyrotroph embryonic factor (Tef), and hepatic leukemia factor (Hlf) [Dbp and Per promoters [Sickness behavior syndrome (SBS) with fatigue and depression is observed in states of immune activation in the course of infections and autoimmune diseases . SBS canor (Hlf) . In NIH-romoters . Interferomoters , 9. TGFβPer and Cry genes in the first three hours before exerting its inhibitory effects in the following hours, it was thought that TNF triggers the well described negative feedback loop by which PER-CRY inhibit their own transcription. Experiments using Per1,Per2-/- and Cry1,Cry2-/- mouse embryonic fibroblasts did not confirm this hypothesis [Per2, Per3, and Dbp, which were decreased in Cirbp-depleted cells [Cirbp expression was only responsible for the effect of TNF when using suboptimal TNF concentrations in the NIH-3T3 cell assay. Taken collectively the pathways involved in TNF mediated abnormal clock gene expression are still unknown. This contrasts the inhibitory effect of TGFβ on clock genes, which results from the induction of Dec1 [The mechanisms involved in dysregulation of clock genes by immune activation have just recently been addressed. Because the addition of TNF to NIH-3T3 cells was found to increase pothesis . Anotherpothesis . Among ted cells , 12. TNFed cells . However of Dec1 . This E-Twist 1 and Twist 2. The two members of the basic helix-loop-helix (bHLH) family of proteins have been characterized as transcriptional regulators, which bind to E-boxes [Twist1 is expressed in embryonic stages and is important for early development and osteoblast differentiation [Twist1 is overexpressed in many human cancers and cancer cell lines, thereby driving tumor invasion and metastasis [In the search for factors, which upon induction by TNF interfere with E-box mediated transcription we have put our focus on the transcriptional regulators  E-boxes . TWIST2 ntiation , 16. Rectastasis –19. TWIStastasis . Both prtastasis .Clock / Bmal1 induced activation of E-boxes of Period genes and Dbp [Twist1 and Twist2 by TNF, the effect being mediated by NF-κB [Twist1 and Twist2 to inhibit the expression of TNF, which may indicate bidirectional regulatory control of their expression [Twist1 to serve as a transcriptional repressor mediating TNF effects on Period and Dbp genes by interfering with CLOCK induced E-box binding and functions thereof.The property of TNF to suppress the expression of clock genes may involve TWIST1 and TWIST2. This hypothesis is based on (i) the effect of TNF to interact with the  and Dbp , (ii) thby NF-κB , 21. Thepression . Here, w2. For assessing the effect of Twist on clock genes cells were synchronized for 1h with serum deprivation and then treated with TNF , IL-1β  or TGFβ2 (2ng/ml) (Peprotech) for 4h as described recently [The murine fibroblast cell line NIH-3T3 (CRL-1658) was obtained from the American Type Culture Collection. The mouse hippocampal neuronal cell line HT22 was obtained from David Schubert at the Salk Institute . [recently . Time kiTwist1 and a DharmaFECT transfection reagent. Twenty-four hours before transfection, the cells were seeded in 6-well plates . The transfections used siRNA solutions (5 μM) in 1× siRNA buffer (Dharmacon). The transfection reactions were performed according to the manufacturer's instructions. After 48 h, the cells were harvested for mRNA analyses.Knockdown experiments were performed in NIH-3T3 and HT22 using ON-TARGETplus siRNA (Dharmacon) against Twist1 and Twist2 was established by stable transfection of TrueClone pCMV6::Twist1, pCMV6::Twist2 and pCMV6::empty plasmid (Origene) into NIH-3T3 cells with FugeneHD transfection reagent (Promega) according to the manufacturer's instructions. G418 (Promega) was used as selection antibiotics at a concentration of 0.5mg/ml.Overexpression of 18 primer (2.5 μM) and reverse transcriptase (10 U) from the nanoScript 2 reverse transcription kit (PrimerDesign) were used for the first strand synthesis according to the manufacturer's suggestions. The cDNA was stored at −20°C.Total RNA from NIH-3T3 and HT22 cells were obtained with the NucleoSpin RNAII Kit (Macherey-Nagel), including DNase digestion. Total RNA (1 μg), anchored oligo(dT)Twist1, Twist2, Dbp, Per1, Per2 and Per3 were commercially available from PrimerDesign Each of the samples was examined in triplicate and subjected to qRT-PCR using PrecisionPLUS qPCR Mastermix (PrimerDesign). HPRT and eEF1a1 probes were used as internal controls. Relative RNA expression levels were determined by normalizing to the internal controls, and the values were calculated using the comparative Ct method [The qRT-PCR primers for t method .Immunoblots were performed with samples containing total protein (40 μg) and 12% NuPage Bis-Tris or 7% Tris-Acetat polyacrylamide gels (LifeTechnologies). The membranes were probed with rabbit polyclonal anti-PER1 , rabbit polyclonal anti PER2 , rabbit monoclonal anti-PER3 , rabbit polyclonal anti-DBP , rabbit polyclonal anti-Twist1  and rabbit polyclonal anti-TWIST2 antibodies . The secondary antibody was a horseradish peroxidase-linked goat anti-rabbit IgG , and it was detected by the chemiluminescence technique using the Immobilon Western ECL system (Millipore). For a loading control, we used a mouse monoclonal anti-p84 antibody . The secondary antibody was a horseradish peroxidase-linked goat anti-mouse IgG . Densitometric analysis was performed using the ImageJ software and the intensity of the control lanes were set as 1.renilla) as an internal control, and 240.8 μl of transfection reagent for triplicates . At 48 h after transfection, cells were washed with PBS and lysed with passive lysis buffer (Promega) to perform a conventional dual luciferase assay (DLR). Lysate (20 μl) was placed into each well of a white 96-well plate and measured with an Infinite M200 pro Luminometer (Tecan). The DLR was performed according to the manufacturer's instructions, except that the amount of substrate was reduced to 50 μl per aliquot. In this assay we tested first a construct containing 3 E-boxes from the Period1 promoter (TTTAGCCACGTGACAGTGTAAGCACACGTGGGCCCTCAAGTCCACGTGCAGGGA), as well as the mutated counterpart (TTTAGCACCGGTACAGTGTAAGCAACCGGTGGCCCTCAAGTCACCGGTCAGGGA). From the Dbp promoter one E-box, wild type (CACGTCCCATGTGGCC) and mutated (CACGTCGCAGTTGGCC) was cloned into the reporter plasmid according to: [Period1 E-Box, as well as the mutated sequence were inserted into the pGL3 promoter vector via the Nhe1/Xho1 site. The cloned sequence consists of three E-boxes within 2.0 kb of the 5’-flanking region of the mouse Per1 gene. The Dbp E-Box and the mutated counterpart were amplified out of a shuttle plasmid and ligated into the BglII restriction site of the pGL3 promoter vector.NIH-3T3 cells were grown as described above. The transfections were performed with Effectene transfection reagent (Qiagen) according to the manufacturer's protocol. Briefly, 24 h before transfection, cells were seeded in 12-well plates . The transfections required DNA (450 ng), the pGL3 promoter vector (firefly) with our inserts, the pRL SV40 vector  according to the manufacturer’s instructions. Chromatin was precipitated with either a monoclonal ChIP grade antibody against TWIST1  or anti CLOCK antibody (ab 3517). As positive control a monoclonal RNA Pol II antibody, as negative control a IgG antibody was used 10μg of sheared chromatin and 2μg of antibody was used in 100μl reaction volume. Eluted and reverse cross-linked chromatin was analyzed via qPCR of region of interest. Primers for E-box regions:Per1 E-box:fw: 5’-GGGTAGTTTCCCTCCCTCAC-3’rv: 5’- TGGCATCTGATTGGCTACTG-3’.Dbp E-box:fw: 5’-TCAACATGGTACAGCCCAGA-3’rv: 5’-TGTGGGAGCTGAGCACATAG-3’.The analysis method entailed solving for the DNA quantity of the ChIP and IgG samples, then calculating the fold enrichment of the ChIP sample relative to the IgG sample.t-test. For grouped measurements with one variable for the selected genes, an one-way ANOVA was used, followed by a Bonferroni post-hoc test to decrease the likelihood of a rare event in multiple comparisons. In the case of two variables (i.e. cytokine treatment and siRNA/overexpression) a two-way ANOVA was used to measure the statistical significance, also followed by a Bonferroni post-hoc test to decrease the α-error margin. P values less than 0.05 were considered significant, 0.01 in ANOVAs with post-hoc tests. +/- SEM is shown by error bars.All experiments were performed in triplets and analyzed with GraphPad Prism (GraphPad Software). To assess the statistical significance between single series of measurements (i.e. untreated vs. treated) we used the two-tailed Student’s Twist genes in various types of cells [Twist1 cells were treated for 4 h with TNF and IL-1β at various concentrations. Both TNF and IL-1β induced the expression of Twist1 in a dose dependent fashion  reporter in the presence or absence of siRNA against Twist1. TNF down-regulates the activation of Per1-3xE-box-luc . In the absence of TNF, CLOCK is 243-fold enriched at the Dbp E-box. The knock-down of Twist1 by siRNA treatment abrogates the enrichment of TWIST1 to the Dbp E-box (22-fold) and despite of the presence of TNF enhanced the binding of CLOCK (221-fold) to the E-box  (Twist1 expression by siRNA increased CLOCK binding at the Per1 E-box despite of the presence of TNF.To test whether the E-box of he E-box . The ove 8-fold) . In the Taken together these results support the hypothesis that TWIST1 induction by TNF competes with CLOCK binding to the E-box.in vitro, and in the liver and SCN of mice with CD40 mediated inflammatory syndrome or upon treatment with lipopolysaccharide (LPS) from gram-negative bacteria, interferon alpha (IFN) or TNF [Per1, Per2, Per3 and Dbp. Upon treatment with TNF and IL-1β NIH-3T3 fibroblasts and HT22 hippocampal neuronal cells show a striking upregulation of Twist1 and Twist2. These data are in agreement with recent reports showing TNF and IL-1β to induce both DNA binding proteins in different types of cells including fibroblasts, macrophages, breast epithelial cells, and various tumor cells [Twist1 expression [Twist1 induction on IFN induced production of Axl. This member of the Tyro 3 kinase family is preferentially expressed in myeloid, neuronal and reproductive system cells. Both the delayed kinetics of Twist1 induction by IFNα and the cell specificity of Axl expression may explain absence of inhibition of clock gene expression when treating NIH-3T3 cells for 4 hr with IFNα.Dysregulation of clock gene expression by proinflammatory cytokines has been described in fibroblasts, human pancreatic cancer cells, and in leukocytes ) or TNF –34. The or cells , 26. BesTwist1 is involved in development and osteoblast differentiation and has been characterized as an essential player in cancer stem cell generation, tumor invasion and metastasis. The data provided here show Twist1 to act as a repressor of the transcriptional activity of CLOCK on E-box mediated induction of Per1 and Dbp expression. siRNA against Twist1 leads to an almost complete prevention from TNF and IL-1β induced inhibition of clock genes. This effect is not shared with effects of siRNA against Twist2. Likewise the overexpression of Twist1, but not of Twist2 inhibits clock gene expression in NIH-3T3 cells. Thus Twist1 is characterized here as the main TNF-induced repressor of clock genes in NIH-3T3 cells. Recently the effect of TNF was shown to be dependent on down-regulation of Cirbp [Cirbp show that downregulation of Cirbp plays only a role in inhibition of clock gene expression at low doses (1 ng/ml) of TNF. At high doses (10 ng/ml) TNF operates in a Cirbp independent way [Twist1.of Cirbp , which iof Cirbp , 13. HowPer1 and Dbp promoter to be associated with absence of CLOCK protein at the respective sites. These data suggest that by occupying the E-box elements in the Per1 and Dbp promoters TWIST1 inhibits binding of CLOCK. It remains to be seen whether Twist1 functions as a homodimer or, as reported previously, the effects of Twist1 may depend on formation of heterodimers with e.g. other class B bHLH proteins [Many of the target genes of TWIST1 and TWIST2 have multiple E-boxes in their promoters. Induction of transcriptional inhibition by TWIST1 includes blocking of DNA binding and/or the action of other transcriptional activators, binding E-boxes as heterodimers with E-proteins, and recruitment of histone deacetylases . In TNF proteins . Moreoveproteins . But genproteins , 37.Period and of the PAR bZip genes Dbp, Tef, and Hlf, but not of Bmal1 and Clock [Twist (see above). In contrast to TNF and IL-1β, TGFβ2 does not activate Twist1 expression. In line with absence of Twist1 activation we find siRNA against Twist1 not to be protective for the effect of TGFβ2 to inhibit clock gene expression. Previous work showed TGFβ to act on the circadian system by induction of Dec1 [Dec1, another bHLH factor, is induced by CLOCK:BMAL1 heterodimer via the CACGTG E-box in the promoter and, thereafter, suppresses transcription by competing with CLOCK-BMAL1 for the DNA binding. DEC1 upregulation by TGFβ resets the circadian phase of peripheral clocks [While TGFβ2 shares with TNF and IL-1β its effect to inhibit the expression of nd Clock , the int of Dec1 . Dec1, al clocks . The difTwist1 may be increased in the periphery and / or in the brain in states of immune activation in infectious–and autoimmune diseases. Upregulated Twist1 may lead to low levels of E-box dependent transcription induced by CLOCK-BMAL1. Since E-boxes are very frequent in the mammalian genome, CLOCK-BMAL1 heterodimers regulate the transcription of many genes besides the clock genes Per and Cry. Thus the TNF mediated increase of Twist1 may modulate a multitude of physiological processes. Experimental in-vivo evidence in support of the hypothesis would require mice with a deletion of the Twist1 gene. However, Twist1-/- mice die at E10.5 with increased apoptosis in multiple tissues [Twist1 lead to the Saethre-Chotzen syndrome, which is associated with severe abnormalities in fusion of the cranial bones and digit defects [Twist1 gene are required to substantiate the hypothesis that Twist1 transmits signals of the immune system to the clock gene network at the transcriptional level.Being induced by proinflammatory cytokines,  tissues . Mutatio defects . Thus cePeriod genes and Dbp points to a previously unrecognized function of Twist1 and suggests that Twist1 plays a critical role linking the immune and clock systems.The ability of TWIST1 to bind to E-box elements and to compete with CLOCK-BMAL1 mediated activation of S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.S5 Dataset(XLSX)Click here for additional data file.S6 Dataset(XLSX)Click here for additional data file.


To establish the key insulin receptor substrate 1 (IRS-1) structural elements required in this insulin regulatory pathway, we investigated the effects of substituting alanine for serine 307 in IRS-1 on the ability of tumor necrosis factor-α (TNF-α) and a related mediator, suppressor of cytokine signaling 3 (SOCS3), to phosphorylate IRS-1 and regulate insulin signaling in the rat retinal Müller cell (rMC-1) cell line.Ser307plasmid or a mutated IRS-1Ser307Ala plasmid. Cells were also treated with recombinant TNF-α or SOCS3 to induce increased levels of these proteins.rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose medium and transfected with either normal IRS-1Ser307Ala, TNF-α and SOCS3 failed to phosphorylate IRS-1. Likewise, resulting downstream effects, including changes in phosphorylation of insulin receptorTyr960, antiapoptotic Akt phosphorylation, and proapoptotic cleavage of caspase 3 were also blocked. We also report for the first time that SOCS3 and TNF-α are reciprocally stimulatory leading to a mutual enhancement of levels of both factors, thus forming a potential positive feedback loop that contributes to insulin receptor resistance.In cells with IRS-1Increases in TNF-α and SOCS3 are triggered by high glucose and through reciprocal stimulation of expression of these two factors, which in turn could be major drivers of insulin resistance and related cell death. The demonstration that a single phosphorylation site is key for these pathways suggests that drugs targeted to this site might be effective in protecting against diabetic damage to the retina. Diabetes produces several physiologic and metabolic changes in the retina, many of which are still poorly understood. One of the first cell types to be altered in response to high glucose is the Müller cell . The expTyr960), which inhibits the interaction between insulin receptor and IRS-1 [In addition to regulating IRS-1, TNF-α can also regulate insulin signal transduction through increasing levels of suppressor of cytokine signaling 3 (SOCS3) . SOCS3 ind IRS-1 . In addind IRS-1 . It is uSince TNF-α and SOCS3 can negatively regulate insulin receptor signaling through IRS-1 in retinal endothelial cells , we wantRat retinal Müller cells  were grown in 5 mM or 25 mM glucose Dulbecco's Modified Eagle Medium . We chose to use this model as we have previously published the effects of β-adrenergic receptor agonists on insulin signaling in these cells . Medium Ser307Ala genes in rMC-1 cells, the cells were passaged and cultured to reach 80% confluency. Cells were then transiently transfected with plasmids expressing either wild-type IRS-1 or mutant IRS-1 (Ser307Ala) genes using GeneMute transfection reagent  according to the manufacturer’s instructions.Some cells were treated with TNF-α  for 30 min, while additional cells were treated with recombinant SOCS3  for 60 min. For the treatments with plasmids, plasmid transfection preceded treatment with SOCS3 or TNF-α. For overexpression of normal IRS-1 and its mutant IRS-1Ser307Ala mutant plasmid as described below. The native and mutant rIRS-1 genes were then sub-cloned in mammalian expression vector pCMV-M1  by double digestion with SacI and HindIII , gel purification of the digestion fragments followed by ligation with T4 DNA ligase .Rat IRS-1 plasmid obtained from Addgene  was used to generate the rIRS-1PfuUltra high-fidelity DNA polymerase (Stratagene) was used for PCR cycling under the following conditions 95 °C for 1 min followed by 18 cycles each consisting of 95 °C for 50 s, 60 °C for 50 s and 68 °C for 1 min per kb of plasmid length. Finally, 7 min at 68 °C was allowed for final extension. The PCR product was then digested with Dpn-I to remove the methylated parental DNA. The nicked vector DNA incorporating the desired mutations was then transformed into XL10-Gold ultracompetent cells followed by plating on selective Luria broth (LB) plates. Mutation was verified with DNA sequencing, and protein levels were confirmed with enzyme-linked immunosorbent assay (ELISA).Mutagenesis was performed using the QuickChange II XL site-directed mutagenesis kit . Briefly, a pair of primers with the desired mutations was designed using Stratagene’s primer design software in such a way that they introduce an alanine instead of serine at the 307 position of the IRS-1 gene while using the plasmid as a template in thermal cycling. PfuUltra high-fidelity from Stratagene). The PCR product was gel purified and cloned in TOPO vector pcDNA 3.2. The cloning reaction was then used to transform One Shot TOP10 chemically competent E. coli, and the bacterial culture was spread on the prewarmed selective plate. Expression plasmids were isolated from single colonies, and the plasmids were analyzed to confirm the presence and correct orientation of the insert with restriction enzyme digestion and DNA sequencing.Rat IRS-1 plasmid and the respective mutant plasmid were generated by sub-cloning the genes from cloning vectors into mammalian expression vectors following the instructions of Invitrogen’s pcDNA Gateway Directional TOPO expression kit . Briefly, forward PCR primers were designed with CACC at the 5′ end of the primer followed by a Kozak translation initiation sequence ATG. Reverse primers were designed conventionally with special care not to include a sequence complementary to the overhang sequence GTGG at the 5′ end. A blunt-end PCR product was produced using a thermostable proofreading DNA polymerase , rMC-1 cells were treated with lysis buffer containing protease and phosphatase inhibitors and scraped into the tubes. Equal amounts of protein from cell lysates were separated on the precast tris-glycine gel (Invitrogen), and blotted onto a nitrocellulose membrane. After blocking in TBST  and 5% (w/v) bovine albumin serum (BSA), the membrane was treated with appropriate primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected with the chemiluminescence reagent kit . Primary antibodies used were phosphorylated Akt (Serine 473), Akt, total IRS-1, SOCS3 , beta actin (Santa Cruz), and insulin receptor . Western blot analyses of proteins of interest were compared to beta actin levels, and a ratio is presented.After appropriate treatments and rinsing with cold PBS PBS .A cleaved caspase 3 ELISA  was used to measure levels of the active apoptotic marker in rMC-1 cells, while phosphorylation of IRS-1 on serine 307 was measured with ELISA  with equal protein loaded (50 μg) for both ELISAs to allow for analyses using optical density measurements. By loading equal protein into the cleaved caspase 3 and IRS-1Statistical significance was determined using a one-way ANOVA with Student Newman Keul’s post-hoc test with p<0.05 taken as statistically significant. Data are shown as means± standard error of the mean (SEM). For western blots, a representative blot is provided.rMC-1 cells used for these studies express key markers of retinal Müller cells, we performed western blotting for two key Müller cell marker proteins, glial fibrillary acidic protein (GFAP) and cellular retinaldehyde-binding protein (CRALBP), as reported in the original description of this cell line in 1998 [To confirm that the  in 1998   and high glucose (HG) led to a significant increase in TNF-α levels with data that demonstrate exogenous TNF-α treatment increased TNF-α levels in rMC-1 cells by approximately 3.5 fold, measured with ELISA. Western blot confirmed that transfection of the normal plasmid and the mutant plasmid resulted in increased levels of total IRS-1 in all groups undergoing transfection or treated with high glucose  TNF-α preferentially phosphorylates IRS-1sduction ,16 and 2sduction . Our newTyr960, which inhibits insulin receptor/IRS-1 interaction, and ubiquinitization of IRS-1, which leads to IRS-1 destruction [Tyr960 in rMC-1 cells grown in high glucose, and this leads to a concomitant decrease in antiapoptotic Akt phosphorylation and an increase in proapoptotic cleavage of caspase 3. In addition, we report for the first time that IRS-1Ser307 is required for SOCS3 actions on IRTyr960, as this does not occur if IRS-1Ser307 is mutated. Thus, TNF-α and SOCS3 require an active serine 307 phosphorylation site on IRS-1 to bring about insulin resistance.A somewhat unexpected finding from these experiments is the high degree to which SOCS3 partners with TNF-α in affecting high-glucose stress responses in rMC-1 cells. It has been previously shown that SOCS3 inhibits insulin signaling through multiple pathways, including phosphorylation of IRtruction ,9,17. OuAnother novel observation in this study is that SOCS3 can increase TNF-α levels. We find that rMC-1 cells in high glucose have increased TNF-α, which is further stimulated with the addition of exogenous SOCS3. These results are consistent with the report that silencing SOCS3 inhibits TNF-α levels in preadipocytes . The recSer307. Increases in TNF-α and SOCS3 are triggered by high glucose and through reciprocal stimulation of the expression of these two factors, which in turn could be major drivers of insulin resistance and related cell death. The demonstration that a single phosphorylation site is key for these pathways suggests that drugs targeted to this site might be effective in protecting against diabetic damage to the retina.In conclusion, TNF-α and SOCS3 inhibit insulin signaling and stimulate retinal rMC-1 cell apoptosis; both mechanisms act through phosphorylation of IRS-1


The following information is missing from the Funding section: This work is also supported in part by UC Davis RISE program and U.S. National Science Foundation through grants IIS-1528203 and IIS-1320229.


Hydrophis cyanocinctus venom gland T7 phage display library, was shown to exhibit significant anti-inflammatory activity in vitro and in vivo. As a TNFR1 antagonist, it reduced cytotoxicity mediated by TNF-α in L929 fibroblasts and effectively inhibited the combination between TNF-α with TNFR1 in surface plasmon resonance analysis. H-SN1 was also shown to suppress TNFR1–associated signaling pathways as it minimized TNF-α-induced NF-кB and MAPK activation in HEK293 embryonic kidney and HT29 adenocarcinoma cell lines. We next determined the effect of H-SN1 in vivo using a murine model of acute colitis induced by dextran sodium sulfate, demonstrating that H-SN1 lowered the clinical parameters of acute colitis including the disease activity index and histologic scores. H-SN1 also inhibited TNF/TNFR1 downstream targets at both mRNA and protein levels. These results indicate that H-SN1 might represent a suitable candidate for use in the treatment of TNF-α-associated inflammatory diseases such as inflammatory bowel diseases.Snake has been used for centuries as a traditional Chinese medicine, especially for therapeutic treatment for inflammatory diseases; however, its mechanisms of action and active constituents remain controversial. In our study, a tumor necrosis factor receptor 1 (TNFR1) selective binding peptide, Hydrostatin-SN1 (H-SN1), which was screened from a  Snake has been used for centuries as a traditional Chinese medicine; however, its active constituents and mechanisms remain largely unknown. Research has demonstrated that snake venom is a mixture of proteins and peptides with various different biological activitiesHydrophis cyanocinctus venom gland T7 phage display library.Tumor necrosis factor  is a highly pleiotropic cytokine that is involved in various autoimmune and inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis and septic shock4578Inflammatory bowel diseases (IBDs) comprise a series of chronic inflammatory diseases that seriously affect the gastrointestinal tract; these have been mainly subdivided as Crohn’s disease and ulcerative colitis. Patients suffering from IBD undergo a group of clinical symptoms including diarrhea, rectal bleeding, and weight lossAs a pleiotropic cytokine, the upregulation of TNF-α expression is a hallmark of inflammatory response. Accordingly, although the etiology of Crohn’s disease remains unclear, a series of research have indicated that TNF-α functions as a core role in the inflammatory response in this disease. Notably, the level of TNF-α is increased in the stool and serum of patients suffering from Crohn’s disease11Hydrophis cyanocinctus venom gland T7 phage display library. To examine the potential anti-inflammatory actions of the screened compounds, the effects of the TNFR1-binding peptide H-SN1 were then determined using the dextran sulfate sodium (DSS) induced colitis murine model. Several correlations between the model and human IBDs have been identified; thus, this model has been considered suitable for investigating the pathogenesis and therapeutic options for IBDsTherefore, to identify likely compounds that would selectively inhibit TNFR1, in our study we selected TNFR1 as the specific target for screening bioactive peptides from a H. cyanocinctus venom gland T7 phage display library was following the guidelines of the OrientExpress cDNA and T7Select System Manuals  as shown in Materials and Methods. The titer of the original library was 1.56 × 106 pfu/ml.The construction of One potential binding peptide was selected after sequencing, named Hydrostatin-SN1, with the nucleotide sequence: 5′-GAC GAA CAA CAC CTA GAG ACC GAA CTA CAC ACT CTC ACC AGC GTG CTG ACA GCC AAT GGA TTC CAA-3′ corresponding to the amino acid sequence: DEQHLETELHTHLTSVLTANGFQ.de-novo prediction of the three dimensional structure of H-SN1 showed a coil-helix-coil conformation . Further competition assays showed that H-SN1 exhibited a marked influence on TNFR1-blockage in a dose-dependent manner (We investigated whether H-SN1 targets TNF-α or inhibits TNF-α binding to TNFR1 using surface plasmon resonance (SPR) analysis. In our study, BIAcore T100 and CM5 sensor chips were utilized. H-SN1 resulted in a dose-dependent resonance when flowed through TNFR1 immobilized on a biosensor chip, demonstrating the direct combination of H-SN1 with TNFR1 . BIAevalt manner .We evaluated the effect of H-SN1 on the biological activity of TNF-α using a TNF-α-mediated L929 cytotoxicity assay15NF-кB and MAPK signaling triggered by the TNF-α/TNFR1 combination is associated with a series of proinflammatory functions of TNF-αTo evaluate the inhibitory chronergy of H-SN1 on TNF-α-mediated NF-κB and MAPK pathway stimulation, 20 ng/ml TNF-α was added to the HEK293 and HT29 cells in the presence of 4 nM H-SN1. As shown in in vivo. Compared with mice treated with DSS alone, mice with administration of H-SN1 presented a significant improvement.The DSS-induced murine colitis model was chosen for the assessment of the anti-inflammatory effects of H-SN1 p < 0.001), but a low concentration of H-SN1 (5 μg/kg) had little effect on colon length reduction.During the entire experiment, the disease activity index (DAI) was monitored each day. As shown in p < 0.01 at 10 μg/kg).Histological injury was also ameliorated by treatment with H-SN1. As shown in TNF-α, as an important proinflammatory mediator, exerts a vital function in colitis. The secretion of TNF-α in turn improves the expression of the cytokine via positive feedback. Therefore, we tested whether the blockage of TNFR1 affects TNF-α expression by disturbing the positive feedback loop. Accordingly, TNF-α expression was significantly raised in the DSS-group , whereasin vivo, we investigated whether treatment with H-SN1 inhibited the NF-κB and MAPK pathways in the colonic tissue of mice suffering from colitis. As shown in To understand the mechanism of H-SN1 function Colonic inflammation consequent to the abnormal activation of immune cells is thought to lead to exorbitant secretion of proinflammatory cytokinesH. cyanocinctus venom gland T7 phage display library, we discovered that H-SN1, a natural peptide, had inhibitory effect on TNF-α induced cytotoxicity in L929 cells dose-dependently, with no significant independent cytotoxicity on these cells with or without actinomycin D. In SPR analysis, we further indicated that H-SN1 could bind to TNFR1 at a KD of 32 μM and markedly suppressed the combination of TNF-α with TNFR1 dose-dependently. These results indicated that H-SN1 could directly bind to TNFR1 and disrupt the combination between TNF-α with its receptor, thereby suppressing its activity. From the data in the article, the H-SN1 has been demonstrated to be a lead peptide and it is potential for the development of new agents for inhibitors of TNFR1 based on the peptide.Through screening a Increasing attention has been focused on the selective inhibition of TNF/TNFR1 axis, which is an alternative to complete blockage of TNF-α causing both TNFRs inhibitionin vivo, the protective effect of H-SN1 in DSS-induced acute colitis in mice was assessed. TNF-α is well known to play a pivotal role in inflammatory response both in experimental colitis and in human IBDs. TNFR1 is an important signaling receptor that mediates the pro-inflammatory function of TNF-α2324To evaluate the anti-inflammatory activity of H-SN1 in vivo suggested that TNF-α might have both direct and indirect effects on the pathogenesis of colitis. The IBD pathogenesis was also correlated with increased cytokine production in the colon. The inflammatory process has been ameliorated using the specific antibodies for neutralization of TNF-α, IL-6, and IL-1β26TNFR1 knockout were shown to exhibit only a trifling mucosal injury and a mild inflammatory cell infiltration in response to DSSFollowing treatment with H-SN1 for 7 days after the attack of colitis, an amelioration in the clinical manifestations and histological signs became apparent, as assessed by the colon length change, DAI and histological scores, and the level of TNF-α. Data from experiments Because H-SN1 is a TNFR1-binding peptide, we also investigated the effect of H-SN1 on TNF-α-mediated NF-κB and MAPK pathway activation in the colonic tissue of mice with colitis. Genes encoding proinflammatory mediators such as IL-6, TNF-αand IL-12 are transcriptionally activated in response to TNF-α, primarily through NF-κB activation32H. cyanocinctus venom gland T7 phage display library, represents a candidate TNFR1 inhibitor that inhibits the TNF-α/TNFR1 interaction and the biological functions of TNF-α in in vitro and in vivo models. Our research found that treatment with H-SN1 exhibited a marked therapeutic effect on the clinical symptoms and histological damage of the inflamed colonic mucosa in DSS-induced murine colitis, and also affects the production of inflammatory mediators  and suppresses inflammatory cell infiltration. Thus, inhibition of the TNF-α-mediated activation of the NF-κB and MAPK pathways might represent important mechanisms by which H-SN1 is able to alleviate colitis in the DSS model. In summary, H-SN1 might serve as a potent candidate for the treatment of TNF-α-associated inflammatory diseases such as IBDs.In conclusion, our investigation demonstrates that H-SN1, a TNFR1-binding peptide screened from a H-SN1 was synthesized and purified up to 95%  and was freshly dissolved in normal saline. TNF-α, TNFR1, and TNFR2 were from Peprotech . DSS was from MPBio .Hydrophis cyanocinctus venom gland T7 phage display library was constructed previously according to the OrientExpress cDNA Manual and the T7Select System Manual (Novagen). The titer of the library was 1.56 × 106 pfu/ml. The Escherichia coli strain BLT5403 was used as the host strain for the phage library.HEK293, HT29, and L929 cell lines were obtained from ATCC. The Hydrophis cyanocinctus venom gland was extracted from fresh venom gland tissues and purified. The T7Select 10-3 OrientExpress cDNA Cloning System, oligo(dT) (Novagen) was chosen to construct the cDNA library. The recombinant vectors were subsequently packaged with T7 Packaging Extracts (Novagen) and propagated in BLT5403. Finally, the titer of the packaged phage library was determined and the amplified library was stored at −80 °CTotal RNA from the 8 phage particles were added into a 96-well plate coated with TNFR1, and then incubated for 1 h at 37 °C. The bound phages were eluted and amplified in E. coli BLT5403, and the titer of the eluted buffer and the amplified phage particles were determined. Individual colonies were picked randomly after three rounds of selection and DNA sequencing and BLAST were used to analyze the inserted sequences.The biopanning procedure comprised three rounds of selection and proceeded following the T7Select System Manual (Novagen). Briefly, 1.0 × 10ab initio predict the three dimensional structure of H-SN1 and the lowest energy structure was identified by PROSA, ERRAT, and RAMPAGE servers.The secondary structure of H-SN1 was predicted using The Protein Structure Prediction (PSIPRED) server. QUARK was used to A molecular docking experiment was used to predict the interaction of H-SN1 with TNFR1 (Protein Database [PDB] code: 1TNR) and analyze the binding regions. CABS-dock was first used to predict the initial complex structure of TNFR1 with H-SN1; RosettaDock was employed for further flexible docking majorization. PyMOL software was chosen to visualize these structures.To evaluate the binding properties of H-SN1, affinity and competition tests were performed by BIAcore . TNFR1 proteins were coupled to a sensor chip after activation with EDC/NHS, and excess activated carboxyl groups were blocked with ethanolamine for 7 min following TNFR1 immobilization. Various concentrations of H-SN1 were injected separately over the chip surface with a flow rate of 10 μl/min in PBS. HCl-Gly buffer (pH 2.0) was used to regenerate the chip between binding cycles. The binding curves were analyzed using BIAevaluation 3.2 software. To investigate whether H-SN1 could block TNF-α/TNFR1 association, 20 nM TNF-α, diluted with or without several concentrations of H-SN1 (TNF-α/H-SN1 = 1:0.5 or 1:5) was allowed to pass through the coupled TNFR1.L929 mouse fibrosarcoma cells were seeded and then incubated for 12 h at 37 °C. Serial concentrations of H-SN1 were dissolved in medium containing 20 ng/ml TNF-α and 0.2 μg/ml actinomycin D, and the TNF-α-free groups were treated with serial dilutions of H-SN1 with or without actinomycin D. The MTT assay was used to determine cytoactivity.Total cell or tissue lysates were extracted with lysis buffer, resolved using 10% SDS-PAGE gel, and transferred to a nitrocellulose filter membrane (0.2 μm). The antigens were detected by specific antibodies followed by the respective secondary antibodies. GAPDH was simultaneously detected to compare the protein loaded into each lane. The following antibodies were used: mouse monoclonal anti-phospho-IκB, mouse monoclonal anti-IκB, rabbit monoclonal anti-ERK, and rabbit monoclonal anti-phospho-ERK ; mouse polyclonal anti-phospho-p38, rabbit polyclonal anti-p38, rabbit monoclonal anti-phospho-JNK, and mouse monoclonal anti-JNK ; and rabbit monoclonal anti-GAPDH was from Santa Cruz Biotechnology .Hydrophis cyanocinctus) was captured in the South China Sea near Guangxi Province (P. R. China). The venom gland was isolated and immediately frozen in liquid nitrogen. BALB/c mice  were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . The Animal Care and Use Committee of the Second Military Medical University approved all the animal tests and experimental protocols including venom gland isolation and the mouse study, which were performed in accordance with the Chinese guidelines for the care and use of laboratory animals.The snake . The normal  and DSS (control) control groups received normal saline at a dose of 10 ml/kg; groups 3 and 4 were administered H-SN1 at 5 and 10 μg/kg, respectively. All treatments were continued for 7 consecutive days. Drugs were given by intraperitoneal injection once daily and were dissolved in normal saline.Formalin-fixed and paraffin-embedded sections of colonic tissues were stained with hematoxylin and eosin. Histological grading was assessed according to previously described criteria 3536.35® Premix Ex Taq™ kit (TaKaRa) to analyze the expression levels of the tested genes. The primers used were designed based on published sequences 18TRIzol Reagent  was used for the extraction of total RNA from colonic tissue specimens. Samples of 1 μg mRNA were reverse-transcribed using the PrimeScript™ RT Master Mix Kit (TaKaRa). Real-time PCR was performed using the SYBRp < 0.05 was considered statistically significant.For multiple comparisons, the data are presented as the means ± SEM and assessed by one-way Analysis of Variance (ANOVA). GraphPad Prism 5 software for Windows was used , How to cite this article: Zheng, Z. et al. Screening of an anti-inflammatory peptide from Hydrophis cyanocinctus and analysis of its activities and mechanism in DSS-induced acute colitis. Sci. Rep.6, 25672; doi: 10.1038/srep25672 (2016).


Normal pressure hydrocephalus (NPH) is a potentially reversible cause of ventriculomegaly characterized by a classic triad of gait disturbance, cognitive impairment and urinary incontinence in older adults. Shunt tube placement is currently the mainstay therapy for NPH. However, unpredictable therapeutic responses as well as associated surgical complications necessitate better characterization of surgical candidates. The purpose of this study was to use MR Elastography (MRE) to determine brain tissue stiffness and outcomes after shunting in NPH patients compared with age and sex matched healthy controls.With IRB approval 10 patients (age range of 67-79 years) with NPH who were scheduled for ventriculoperitoneal shunting underwent preoperative MRE and were correlated with 21 age- and sex-matched normal controls (age range of 67-80 years). Studies were performed on a 3T scanner with a single-shot spin-echo EPI pulse sequence. Shear waves were introduced into the brain through an external source of vibration using a frequency of 60 Hz. We calculated the elasticity of different regions of interest (ROI) in the brain including the whole brain excluding the cerebellum (cerebrum), frontal, temporal, parietal, occipital lobes, deep grey matter/ white matter and the cerebellum. Associations between ROIs and symptoms were evaluated including cognitive decline, urinary incontinence, gait disturbance, duration of symptoms, opening pressure, improvement after lumbar puncture, and postoperative improvement. Statistical analysis included t test and linear regression.MRE demonstrated significantly increased parenchymal stiffness among NPH patients as compared to normal controls in multiple ROIs, including the cerebrum, occipital, and parietal lobes . Postoperative improvement was associated with significant increased stiffness in deep grey/white matter, whereas postoperative failure was associated with significant increased temporal lobe stiffness .Brain MRE of patients with NPH revealed increased stiffness of the cerebrum, occipital and parietal lobes with a significant association between a range of NPH symptoms including the classic clinical triad with increased parenchymal stiffness. Surgically-responsive NPH was significantly associated with deep grey/white stiffness. MRE may guide selection of patients for shunting in the setting of equivocal diagnoses of NPH.