NRAMP1 Research Articles

The Role of NRAMP1/SLC11A1 Gene Variant D543N (1730G/A) in the Genetic Susceptibility to Develop Rheumatoid Arthritis in the Mexican Mestizo population.

Rheumatoid arthritis is a chronic inflammatory disease whose cause has not been fully elucidated. However, genetic factors seem to have an important role in its pathogenesis. We analyzed the possible association between rheumatoid arthritis and variants of the SLC11A1 gene, which encodes for NRAMP1, a protein involved in the activation of phagocytes and synthesis of proinflammatory cytokines. In a case-control study in a Mexican Mestizo population, blood samples from 188 patients with rheumatoid arthritis and 133 healthy individuals were obtained to determine the frequency of SLC11A1 gene variants INT4 (469+14G/C or rs3731865), D543N (1730G/A or rs17235409) and 3'UTR (1729+55del4 or rs17235416) by polymerase chain reaction and restriction fragment length polymorphism. We found similar frequencies of INT4 and 3'UTR polymorphisms in patients and controls (p = 0.18 and 0.89, respectively). In contrast, a significantly lower frequency of the D543N polymorphism was observed in patients with rheumatoid arthritis compared to controls (p corrected = 0.016; OR: 0.48; 95% CI: 0.28-0.80). Our data suggest that the D543N variant of SLC11A1 gene has a protective effect in the development of rheumatoid arthritis, an interesting finding that has not been previously reported in any population.

Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects

BackgroundThe CRISPR-Cas9 system is a widely utilized platform for transgenic animal production in various species, although its off-target effects should be addressed. Several applications of this tool have been proposed in model animals but remain insufficient for transgenic livestock production.ResultsHere, we report the first application of single Cas9 nickase (Cas9n) to induce gene insertion at a selected locus in cattle. We identify the main binding sites of a catalytically inactive Cas9 (dCas9) protein in bovine fetal fibroblast cells (BFFs) with chromatin immunoprecipitation sequencing (ChIP-seq). Subsequently, we demonstrate that a single Cas9n-induced single-strand break can stimulate the insertion of the natural resistance-associated macrophage protein-1 (NRAMP1) gene with reduced, but still considerable, off-target effects. Through somatic cell nuclear transfer, we finally obtain transgenic cattle with increased resistance to tuberculosis.ConclusionsOur results contribute to the development of CRISPR-Cas9 system for agriculture applications.

NRAMPI (SLC11A1) Gene-1703 G/A Polymorphisms as Prognostic and Diagnostic Marker of Rheumatoid Arthritis in Egyptian Population

Rheumatoid arthritis (RA) is an autoimmune disease with difficulty to diagnose. The association of natural resistance-associated macrophage protein-1 (NRAMP-1) gene polymorphisms with rheumatoid arthritis (RA) is the aim of this study. Methods: Two hundred individuals who were divided into two groups: RA patients group (100 Patients with RA) and control group: (100 apparently healthy subjects). NRAMP1 polymorphisms were determined by the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) method, including D543N. Results: Rheumatoid arthritis patients demonstrated significant increase in C-reactive protein (CRP), Rheumatoid Factor Ab, Human cartilage oligomeric matrix protein (COMP) and Anti-Cyclic Citrullinated Peptide (anti-CCP) in comparison to normal. Clinical disease activity index (CDAI) and the radiological erosion score of joints in RA patients were 29.8 ± 5.3 and 60.03 ± 38.71 respectively. The G/G, G/A and A/A genotypes were 64, 33, and 3% respectively in RA patients and were 60, 25, and 15% respectively in controls with a significant difference between RA patients and controls. The CDAI and radiological erosion score of joints had the least values in NN followed by DN while DD patients had the highest levels. The patients with phenotype DD had a grade III of disease in 40 patients and grade IV in 21 patients while grade II found in three patients. However, in patients with phenotype DN, the grading of disease II, III and IV were found to be 10, 20 and 3 respectively. All patients with NN phenotype showed a grade II of disease. There was a significant difference in percentage between RA patients and controls as regard to G or A alleles distribution. The nodule associated with G/G genotype in 58.8% while bone erosion associated with G/G genotype in 65% of RA patients. Conclusion: The present study showed that NRAMP1 1703G (543D) is a risk factor for the development of RA. The estimated NRAMP1 1703G haplotype is associated with susceptibility to RA. In RA patients with 1703A, development of rheumatoid nodule is absent.

Role of hydrogen sulfide in the methyl jasmonate response to cadmium stress in foxtail millet.

Methyl jasmonate (MeJA), a vital cellular regulator, mediates diverse developmental processes and defense responses against environmental stresse. Recently, a novel gasotransmitter, hydrogen sulfide (H2S), was found to have similar functions, but the interactions between H2S and MeJA in the acquisition of cadmium (Cd) tolerance have not been reported. Treating foxtail millet with 1 microM MeJA not only enhanced Cd tolerance and alleviated growth inhibitions but also decreased the contents of hydrogen peroxide, malondialdehyde and Cd in seedlings under 200 microM of Cd stress. Exogenous application of MeJA inhibited the transcript levels of the Natural Resistance-Associated Macrophage Protein (NRAMP1 and NRAMP6) and intensified Cd-induced expression of the homeostasis-related genes (MTP1, MTP12, CAX2 and ZIP4, besides HMA3). In addition, treatment with MeJA induced the production of endogenous H2S. Fumigation with sodium hydrosulfide (H2S donor) significantly enhanced MeJA-induced Cd tolerance, but this ability was weakened when H2S biosynthesis was inhibited with hydroxylamine. These results suggest that pretreatment with MeJA alleviated Cd stress and that this improvement was mediated by H2S in foxtail millet.

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

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

Evidence for gene-gene epistatic interactions between susceptibility genes for Mycobacterium avium subsp. paratuberculosis infection in cattle

Johne's disease is a chronic granulomatous inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), with a particularly negative impact on the economy of the dairy industry. In recent years, several whole genome and candidate gene association studies have been published reporting MAP susceptibility genes, but the putative interaction between them remains unknown. Here, twenty-four single nucleotide polymorphisms in the bovine SLC11A1, NOD2, SP110, TLR2, TLR4, and CD209 genes, previously related with paratuberculosis disease, have been analyzed. Several significant (P<0.05) pair-wise genetic interactions were detected: CD209-TLR4, CD209-TLR2, TLR4-TLR2, SP110-SLC11A1, SP110-TLR2, and SP110-NOD2. The statistical interaction described here between bovine CD209 and TLR4 genes may be indicative of the biological interaction between their protein products upon infection by mycobacteria, as has been reported to occur in humans. Overall, this is the first evidence of epistasis among bovine innate immunity genes affecting susceptibility to MAP infection, corroborating, in turn, their implication in the disease.

Crystal Structure and Conformational Change Mechanism of a Bacterial Nramp-Family Divalent Metal Transporter

The widely conserved natural resistance-associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a (absent from a previous Nramp structure). Mapping our cysteine accessibility scanning results onto this structure, we identified the metal-permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations that impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal-permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, theTM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state.

Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots.

The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants . Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds.

The HvNramp5 Transporter Mediates Uptake of Cadmium and Manganese, But Not Iron.

The Natural Resistance Associated Macrophage Protein (Nramp) represents a transporter family for metal ions in all organisms. Here, we functionally characterized a member of Nramp family in barley (Hordeum vulgare), HvNramp5. This member showed different expression patterns, transport substrate specificity, and cellular localization from its close homolog in rice (Oryza sativa), OsNramp5, although HvNramp5 was also localized to the plasma membrane. HvNramp5 was mainly expressed in the roots and its expression was not affected by Cd and deficiency of Zn, Cu, and Mn, but slightly up-regulated by Fe deficiency. Spatial expression analysis showed that the expression of HvNramp5 was higher in the root tips than that in the basal root regions. Furthermore, analysis with laser microdissection revealed higher expression of HvNramp5 in the outer root cell layers. HvNramp5 showed transport activity for both Mn2+ and Cd2+, but not for Fe2+ when expressed in yeast. Immunostaining with a HvNramp5 antibody showed that this protein was localized in the root epidermal cells without polarity. Knockdown of HvNramp5 in barley resulted in a significant reduction in the seedling growth at low Mn supply, but this reduction was rescued at high Mn supply. The concentration of Mn and Cd, but not other metals including Cu, Zn, and Fe, was decreased in both the roots and shoots of knockdown lines compared with the wild-type barley. These results indicate that HvNramp5 is a transporter required for uptake of Mn and Cd, but not for Fe, and that barley has a distinct uptake system from rice.

Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters

Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur's presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it-more effectively than any other amino acid-increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.

Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea.

Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein-protein interaction.

Sequencing and phylogenetic analysis of the SLC11A1 gene in pigs

ABSTRACTThe solute carrier family 11 member A1 (SLC11A1), also known as natural resistance-associated macrophage protein 1 (NRAMP1), is a divalent transition metal (iron and manganese) transporter involved in iron metabolism and host resistance to certain pathogens. To study the genetic diversity of the SLC11A1 gene in Doom pigs of Assam especially on its evolution and differentiation within and among species, the partial sequence of the SLC11A1 gene was sequenced, characterized and compared with other published sequences of pigs and other species livestock. The gene sequence of Doom pigs showed the highest sequence identity with EF200584.1 (exotic pig) and the lowest similarity AY368475.1 (large white strain 008). One single nucleotide polymorphism (SNP) was identified in the heterozygous sequence at the 736 bp position (A→G). The sequence showed the highest sequence identity (81.74%) with that of O. aries and the lowest similarity (39.12%) with B. bubalis (Mehsana breed), respectively. Phylogenetic analysis revealed that the Doom pig is more closely related to EU135795.1 (Chinese local pig) and EF200584.1 (Pig, Iowa State University). The sequencing and phylogenetic analysis of the SLC11A1 gene of Doom pigs could be used for genetic selection with disease-resistance varieties and upgradation of indigenous germplasm of domestic livestock.

Interactions and accumulation differences of metal(loid)s in three sea cucumber species collected from the Northern Mediterranean Sea

This study was conducted on Holothuria polii, Holothuria tubulosa, and Holothuria mammata collected from five stations with different depths in the Northern Mediterranean Sea. The body walls and guts of these holothurians were examined in terms of interactions of 10 metals (iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), chromium (Cr), cobalt (Co), vanadium (V), nickel (Ni), cadmium (Cd), and lead (Pb)) and one metalloid (arsenic (As)) using a multivariate analysis, and interspecies differences were determined. The multivariate analysis of variance (MANOVA) revealed significant differences between the species in terms of metal(loid) accumulations. The principal component analysis (PCA) showed a more association between H. tubulosa and H. polii with regard to the accumulation. The cluster analysis (CA) located Pb concentrations of the guts to the farthest place from all elements regardless of the species. A correlation analysis displayed that the element concentrations of the guts were more closely related to each other compared with those of the walls. The most inconsistent element in terms of correlations was the gut Fe contents. Accordingly, while Fe concentrations of H. mammata and H. tubulosa were correlated with all elements (except Pb) in divalent metal transporter 1 (DMT1) (divalent cation transporter 1 (DCT1) or natural resistance-associated macrophage protein 2 (NRAMP2)) belonging to the NRAM protein family, this was not the case in H. polii. Consequently, significant relationships between accumulated metal(loid)s that changed by tissues and sea cucumber species were observed.

Resistance of Mice of the 129 Background to Yersinia pestis Maps to Multiple Loci on Chromosome 1.

Yersinia pestis is a Gram-negative bacterium that is the causative agent of bubonic and pneumonic plague. It is commonly acquired by mammals such as rodents and humans via the bite of an infected flea. We previously reported that multiple substrains of the 129 mouse background are resistant to pigmentation locus-negative (pgm(-)) Yersinia pestis and that this phenotype maps to a 30-centimorgan (cM) region located on chromosome 1. In this study, we have further delineated this plague resistance locus to a region of less than 20 cM through the creation and phenotyping of recombinant offspring arising from novel crossovers in this region. Furthermore, our experiments have revealed that there are at least two alleles in this initial locus, both of which are required for resistance on a susceptible C57BL/6 background. These two alleles work in trans since resistance is restored in offspring possessing one allele contributed by each parent. Our studies also indicated that the Slc11a1 gene (formerly known as Nramp1) located within the chromosome1 locus is not responsible for conferring resistance to 129 mice.

Study of the correlation between NRAMP1 gene polymorphisms and susceptibility to tuberculosis in Tibetan people in Qinghai

Objective To investigate the correlation between NRAMP1 gene polymorphisms and susceptibility to tuberculosis (TB) in Tibetan people in Qinghai. Methods A case-control study was conducted in this study, involving 99 Tibetan patients with TB and 89 healthy Tibetans. The single nucleotide polymorphisms of NRAMP1 gene at rs17235409 and rs3731865 sites were detected by using TaqMan probe method. Gene cloning and sequencing typing were performed to analyze the single nucleotide polymorphisms of NRAMP1 gene at the rs17235416 site. SPASS20.0 software was used to statistically analyze the correlation between NRAMP1 gene polymorphisms and susceptibility to TB in Tibetan people. Results No significant difference in the genotype frequencies of rs3731865 and rs17235409 was found between the two groups (χ2=0.852, P=0.356; χ2=0.279, P=0.597). The genotype frequencies of TGTG/TGTG and TGTG/del+ del/del at the rs17235416 site were 70.7% (70/99) and 29.3% (29/99) in patients with TB and 86.5% (77/89) and 13.5% (12/89) in healthy subjects. There were significant differences in the genotype frequencies of TGTG/TGTG and TGTG/del+ del/del between the two groups (χ2=6.870, P=0.009). The genotypes of TGTG/del and del/del at rs17235416 were risk factors for TB (OR=0.376; 95%CI: 0.178-1.794 as compared with the TGTG/TGTG genotype in Tibetan people in Qinghai. Conclusion This study suggested that the NRAMP1 gene polymorphisms at rs3731865 and rs1723409 sites had no correlation with the susceptibility to TB in Tibetans in Qinghai. However, the NRAMP1 gene polymorphisms at rs17235416 site were correlated with the susceptibility to TB. The TGTG/del alleles at the rs17235416 site might be the risk factors for tuberculosis in Tibetans in Qinghai. Key words: Tibetan; NRAMP1 gene polymorphism; Tuberculosis; Macrophage

Natural resistance-associated macrophage protein 1 gene polymorphisms in thalassemia patients with tuberculosis infection

that needs regular blood transfusions leading to accumulation of iron in the cells. This iron overload level in macrophage might cause intracellular bacteria, particularly Mycobacterium tuberculosis (MTB) to multiply. Polymorphisms in natural resistance-associated macrophage protein 1 (NRAMP1), a metal transporter across the phagosome membrane, play important role in regulating iron, which is also needed by MTB. Increased iron in thalassemia patients may have an increased potential risk for TB.Objective To compare natural resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms (INT4, D543N, and 3’UTR) in thalassemia patients with and without tuberculosis (TB) infection.Methods A cross-sectional measurement of NRAMP1 genetic polymorphisms was performed in pediatric thalassemia patients with TB (n=40) and without TB (n=50). Iron status including serum iron, total iron-binding capacity (TIBC), and ferritin, was compared between the two groups. The NRAMP1 genetic polymorphisms were analysed using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Allelic and genotypic distributions of each polymorphism were assessed for possible associations with TB infection.Results Mean serum iron and TIBC in thalassemia patients with TB were higher compared to thalassemia patients without TB (mean serum: 166.26 vs. 134.92 μmol/L, respectively; P=0.026) and (mean TIBC: 236.78 vs. 195.84 μmol/L, respectively; P=0.029). In thalassemia patients with TB, we observed significantly higher frequency of the C allele in INT4 (10% vs. 2%, respectively; OR=5.44; 95%CI 1.1 to 26.4; P=0.02) and the TGTG deletion allele (78.8% vs. 51%, respectively; OR=3.56; 95%CI 1.83 to 6.9; P=0.0002) in 3’UTR polymorphisms than in thalassemia patients without TB. There were no significant differences in distributions of the A allele between TB and non-TB groups (16.3% vs. 15%, respectively; P=0.84) or the GA genotype (32.5% vs. 30%, respectively; P=0.79) in D543N.Conclusion The NRAMP1 polymorphisms are known to be associated with major gene susceptibility to TB, and in our thalassemia patients this association is even more pronounced.

Cadmium-stress mitigation through gene expression of rice and silicon addition

Cadmium (Cd) pollution is one of the major concerns in the development of sustainable agriculture, particularly for rice production. Silicon (Si) was recently recognized for its ability to mitigate a variety of abiotic stresses including that caused by Cd on rice. However, mechanism of the complex process is still not fully understood. Under Cd-stress, the low Si-influx 1-RNAi transgenic Lemont rice (Lsi1-RNAi line) exhibited an increased Cd-uptake over its counterparts, the wild type and the Lsi1-overexpression transgenic rice (Lsi1-OE line). In contrast, the Lsi1 expression-enhanced Lsi1-OE line showed the greatest Si-uptake among the three lines, with the highest activities on anti-oxidants (such as, superoxide dismutase) and the lowest content of malondialdehyde. Lsi1 also displayed a negative regulation on Low Cd gene and the natural resistance-associated macrophage proteins, Nramp5, indicating its capacity to alleviate Cd-stress on rice. The results obtained by this study suggested that the mitigation of Cd-toxicity on rice by Si might involve functions, such as the inhibition on Cd-uptake and transport and the enhancement on anti-oxidative enzyme activities, as well as the Lsi1-related expression on regulation of Si-uptake in rice. A new avenue might become available for overcoming the rampant pollution that threatens the rice production in China.

Macrophage Subsets Within Granulomatous Intestinal Lesions in Bovine Paratuberculosis.

Animals infected with Mycobacterium avium subspecies paratuberculosis show a variety of granulomatous lesions that range from focal forms, seen in the subclinical stages, to diffuse lesions associated with clinical signs. The aim of this study was to phenotypically characterize the macrophages present in the different lesion types using immunohistochemical methods. Lesions from a total of 23 animals with bovine paratuberculosis, natural and experimental, were examined by immunohistochemistry. Antibodies against inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), CD163, interleukin 10 (IL-10), transforming growth factor β (TGF-β), major histocompatibility complex (MHC) class II, natural resistance-associated macrophage protein 1 (Nramp-1), calprotectin, Ki-67, CD68, lysozyme, and ionized calcium-binding adaptor molecule 1 (Iba-1) molecules were employed. Samples were scored semiquantitatively using a complete histological score (H-score), reflecting the staining intensity and the percentage of immunolabeled macrophages. Differences in the H-score were seen depending on the lesion type. In focal lesions, with none or few acid-fast bacilli (AFB), macrophages were polarized toward M1 phenotype, with high H-scores for iNOS and TNF-α. Diffuse multibacillary lesions showed M2 differentiation, with high expression of CD163, IL-10, and TGF-β as well as Nramp-1 and MHC class II antigens. Macrophages in diffuse paucibacillary forms showed high H-scores for iNOS but low ones for TNF-α. Diffuse lesions, either multibacillary or paucibacillary, showed high calprotectin and low Ki-67 expression, suggesting a progressive character, while focal forms, with low H-scores for these antigens, would be consistent with latency. Lysozyme and CD68 expression were related to the amount of AFB. H-score for Iba-1 antibody was similar among all types. The findings of this study provide insights into the polarization status of macrophages and lesion development in bovine paratuberculosis.

MBL, P2X7, and SLC11A1 gene polymorphisms in patients with oropharyngeal tularemia

Conclusion: A significant association was found of oropharyngeal tularemia with SLC11A1 allele polymorphism (INT4 G/C) and MBL2 C + 4T (P/Q). These results indicate C allele and Q allele might be a risk factor for the development of oropharyngeal tularemia.Aim: This study aimed to investigate the relationship of SLC11A1, MBL, and P2X7 gene polymorphism with oropharyngeal tularemia.Methods: The study included totally 120 patients who were diagnosed with oropharyngeal tularemia. Frequencies of polymorphisms in the following genes were analyzed both in the patient and control groups in the study: SLC11A1 (5’(GT)n Allele 2/3, Int4 G/C, 3’ UTR, D543N G/A), MBL (MBL2 C + 4T (P/Q), and P2X7 (−762 C/T and 1513 A/C).Results: Among all polymorphisms that were investigated in this study, SLC11A1 gene showed a significance in the distriburtion of polymorphism allelle frequency at the INT4 region. Frequency of C allele was 54 (28%) in patients with oropharyngeal tularemia, and 31 (13%) in the control group (p = 0.006 and OR = 1.96 (1.21–3.20)). An association was detected between MBL2 C + 4T (P/Q) gene polymorphism and oropharyngeal tularemia (p < 0.005 and OR = 0.30 (0.19–0.48)). No significant relation was found between P2X7 (−762 C/T and 1513 A/C) gene polymorphism and oropharyngeal tularemia in this study (p > 0.05).

Functional SLC11A1 (NRAMP1) enhances protection from Coxiella burnetii infection

Abstract SLC11A1 is a divalent ion transporter formerly known as the natural resistance-associated macrophage protein (NRAMP1). Also known as the Bcg/Lsh/Ity locus, SLC11A1 was originally characterized as the resistance factor for Mycobacterium, Leshmania, and Salmonella infections. This protein is non-functional in C57BL/6 mice, thus, the susceptibility of this strain to these pathogens. We recently characterized the expression of SLC11A1 in the majority of γδ T cells and NK cells, which are primary sources of innate IFN-γ. Cells expressing SLC11A1 were more prone to activation. We hypothesized that SLC11A1 would have an important function in infections in which IFN-γ was a critical component. Only two genes have been described as potential susceptibility loci for Q fever caused by Coxiella burnetii infection, they are IFN-γ and SLC11A1 in humans. Thus, we assessed C. burnetii infection in congenic mice with and without functional SLC11A1. SLC11A1+ mice had improved disease symptoms, harbored fewer bacteria, and had increased expression of innate cytokines, including IFN-γ. The results indicate that mice expressing functional SLC11A1 were more resistant to infection with C. burnetii, suggesting an expanded role for this protein in innate immune protection.