Background: Iron acquisition is essential for the growth of Mycobacterium tuberculosis. Hepcidin is known as an antimicrobial peptide and a component of the innate immune response. Hepcidin inhibits M. tuberculosis growth in vitro. In this study, we decided to identify-582A> G variants of the HAMP promoter in patients with tuberculosis (TB) and investigate its effect on serum iron, ferritin, and hepcidin levels. Methods: The sample population consisted of 105 patients with TB and 104 healthy individuals. The-582A> G polymorphism was genotyped using a tetra-primers PCR set. Serum levels of hepcidin were determined using an ELISA kit. Statistical analysis was performed using SPSS software. Results: The G allele is meaningfully associated with TB disease (95% confidence interval = 2-4.8, p G polymorphism genotypes. There was significant reverse correlation between hepcidin and iron (r =-0.849, p = 0.006). Conclusion: A high association was found between serum hepcidin levels and the HAMP-582A> G variants in patients with TB. These observations indicate a hypothetical role of this polymorphism in iron metabolism. Hepcidin could perhaps be an option for the treatment of TB. © 2014 Informa Healthcare

Ajami, A.

Farazmandfar, T.

Javaheri-Kermani, M.

Yazdani, Y.

Mina Javaheri-Kermani

Touraj Farazmandfar

Abolghasem Ajami

Yaghoub Yazdani

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Scandinavian Journal of Infectious Diseases

Impact of hepcidin antimicrobial peptide on iron overload in tuberculosis patients

Golestan University of Medical Sciences Repository

 Correspondence: Yaghoub Yazdani, PhD, Assistant Professor of Immunology, Department of Molecular Medicine, Advanced Medical Science Technologies, Golestan University of Medical Sciences, Gorgan, Iran. Tel:    981714425995. PO Box: 4918936316. E-mail: yazdani@goums.ac.ir  (Received  15  April  2014 ; accepted  21  May  2014 ) Scandinavian Journal of Infectious Diseases, 2014; 46: 693–696ISSN 0036-5548 print/ISSN 1651-1980 online © 2014 Informa HealthcareDOI: 10.3109/00365548.2014.929736 ORIGINAL ARTICLE  Impact of hepcidin antimicrobial peptide on iron overload in tuberculosis patients  MINA  JAVAHERI-KERMANI 1 ,  TOURAJ  FARAZMANDFAR 1,2 ,  ABOLGHASEM  AJAMI 2  &  YAGHOUB  YAZDANI 1  1 Infectious Diseases Research Center and Laboratory Science Research Center, Golestan University of Medical Sciences, Gorgan, Iran and  2 Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran  Abstract  Background: Iron acquisition is essential for the growth of Mycobacterium tuberculosis. Hepcidin is known as an antimicrobial peptide and a component of the innate immune response. Hepcidin inhibits M. tuberculosis growth in vitro. In this study, we decided to identify   582A   G variants of the  HAMP promoter in patients with tuberculosis (TB) and investigate its effect on serum iron, ferritin, and hepcidin levels. Methods: The sample population consisted of 105 patients with TB and 104 healthy individuals. The   582A   G polymorphism was genotyped using a tetra-primers PCR set. Serum levels of hepcidin were determined using an ELISA kit. Statistical analysis was performed using SPSS software.  Results: The G allele is meaningfully associated with TB disease (95% confi dence interval    2 – 4.8,  p   0.000). Signifi cant differences were seen in the levels of serum iron and hepcidin but not ferritin between the   582A  G polymorphism genotypes. There was signifi cant reverse correlation between hepcidin and iron ( r     0.849,  p    0.006).  Conclusion: A high association was found between serum hepcidin levels and the  HAMP   582A   G variants in patients with TB. These observations indicate a hypothetical role of this polymorphism in iron metabolism. Hepcidin could perhaps be an option for the treatment of TB.   Keywords:  Hepcidin ,  polymorphism ,  iron ,  tuberculosis  Introduction  Tuberculosis (TB) is one of the most critical infec-tious diseases in the world. In 2012, it had an incidence about 9.4 million cases and 1.3 million people died from TB. One-third of the worldwide population is generally thought to be latently infected [1], although only 10% of infected individuals develop TB [2]. Iron acquisition is essential for the growth of Mycobacterium tuberculosis (MTB) [3].  Hepcidin is known as an antimicrobial peptide and a component of the innate immune response, and it inhibits MTB growth in vitro [4]. Moreover, this molecule affects a variety of physiological pro-cesses and also functions as a signaling mediator in host defense and infl ammation [5]. The hepcidin antimicrobial peptide ( HAMP ) gene is mainly expressed in the liver in iron overload states [6]. Hepcidin regulates iron homeostasis by decreasing iron content of blood through inhibition of intestinal iron absorption and macrophage iron release [7 – 10]. Hepcidin binds ferroportin, a mammalian iron exporter, and causes its internalization and degrada-tion, resulting in the inhibition of iron fl ow [11]. Ferroportin is expressed highly on duodenal entero-cytes and macrophages [12]. When hepcidin levels increase, both dietary iron uptake and recycling iron resulting from decomposition of senescent red cells by macrophages are decreased. MTB in human monocyte-derived macrophages can acquire iron bound to ferritin, transferrin, and citrate [13]; therefore, high hepcidin levels lead to the reduced availability of iron for MTB. Anemia is a common complication of high hepcidin levels due to the reduced iron supply for erythropoiesis in bone mar-row [14]. Iron overload and infl ammation stimulate hepcidin expression. Hepcidin synthesis is also suppressed by anemia and hypoxia [15,16]. 694 M. Javaheri-Kermani et al.  Review of previous studies indicates that   582A   G variants are associated with hepcidin promoter activity [17 – 19] and, therefore, may play a role in iron overload. In this study, we decided to identify   582A   G variants of the  HAMP promoter in TB patients and investigate their effect on serum iron, ferritin, and hepcidin levels.  Material and methods  Sample preparation  The sample population of this study consisted of 105 patients with TB and 104 healthy individuals who had not received regular iron chelation therapy for several months. They were recruited from medical centers of Golestan province in northern Iran. Infor-mation on the patients ’ serum iron and ferritin levels was extracted from medical records. Blood samples were collected, and genomic DNA was extracted from whole blood using a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany).  Genotyping of   582A   G variants  The rs10421768 single nucleotide polymorphism located in 582 base pairs (bp) upstream translation initiation codon was genotyped by using a tetra-primers PCR set. Primer design was performed using Gene Runner software (version 3.05; Hastings, USA) and  HAMP gene sequence information (GenBank accession no. NG_011563) (Table I). PCR was performed using Taq DNA Polymerase 2x Master Mix RED (Ampli-qon, Copenhagen, Denmark) with 50 – 100 ng of DNA and 5 pmol of each primer on a thermal cycler (Bio-Rad, Munich, Germany). PCR conditions included one step initial denaturation (95 ° C for 10 min), 35 cycles (95 ° C for 30 s, 60 ° C for 30 s, and 72 ° C for 30 s), and a fi nal extension at 72 ° C for 7 min. Following ampli-fi cation, PCR products were electrophoresed in a 2% agarose gel (Fermentas, Sankt Leon-Rot, Germany).  Hepcidin ELISA assay  Serum samples were collected from patients by centrifugation of blood samples at 1500  g for 10 min. Serum levels of hepcidin were determined using a commercial ELISA kit (DRG, GmbH, Marburg, Germany) according to the manufacturer ’ s protocol. The fi nal solution color was photometrically mea-sured at 450/630 nm wavelength with a microplate reader (Bio-Rad). The 5 – 95% range in normal healthy adults is 13.3 – 54.4 ng/ml according to the manufacturer.  Statistical analysis  Statistical analysis was performed using SPSS soft-ware (version 17.0; Chicago, USA). The frequencies of associations between genotypes and disease were analyzed using chi-squared and Fisher ’ s exact tests. The differences seen in the values of the biochemical indicators among the   582A   G genotypes were tested by the Kruskal – Wallis test. The relationship between variables was estimated by Pearson correla-tion analysis. A  p value    0.05 was considered signifi cant.  Results  The alleles were determined by product size band; 317 bp for the A allele, 150 bp for G allele, and 440 bp for control, as shown in Figure 1.  The genotype and allele frequencies of the TB patient group and control group are demonstrated in Table II. The wild-type genotype (AA) was observed in 21 (20%) of the patients, whereas 72 (68.6%) were heterozygous (GA) and 12 (11.4%) were homozygous (GG). The control group included 66 (63.5%) individuals with AA, 32 (30.8%) with GA, and 6 (5.7%) with GG. There were highly sig-nifi cant differences regarding   582A   G polymor-phism of  HAMP between TB patients and the control group (Table II). The results indicate that G allele is meaningfully associated with the TB disease (95% confi dence interval [95% CI]    2 – 4.8,  p    0.000). The  HAMP   582A   G genotypes in TB patients and the control group were in accordance with Hardy – Weinberg equilibrium.  Signifi cant differences were seen in the serum iron and hepcidin levels in   582A   G polymorphism  Table I. Primers used for   582A   G polymorphism determination and sequence analysis. Description Primer Sequence (5 ′ → 3 ′ )  582 A   G Forward outer CTTAAGCGATCTGCCTCAGReverse outer AGGAGTGTCTGGCATGTTGForward inner (G) GGTCTGACACTGGGAAAACAGCGReverse inner (A) GTGTGCCCGATCCGCCCGTSequence analysis Forward TGATATCCCAAAGAAGAGTAGCReverse CAACTTTCCTGGCAACCTC  Hepcidin promoter polymorphism in tuberculosis   695genotypes (Table III). These results indicate that iron levels in GA and GG genotypes have higher values ( p    0.003). Unlike iron, hepcidin concentration decreased in GA and GG genotypes ( p    0.030). Based on the results in Table IV, there was signifi -cant reverse correlation between hepcidin and iron ( r     0.849,  p    0.006) and no signifi cant cor-relation between hepcidin and ferritin ( r    0.151,  p    0.341).  Discussion  MTB as a cause of TB disease requires iron for growth and replication [3]. Hepcidin is an antimicro-bial peptide that regulates iron homeostasis [20,21], and better understanding of its role in TB disease may be interesting. With regard to the points men-tioned, there are not many studies on the relationship of hepcidin to TB disease development [4,22]. In this work, we genotyped   582A   G polymorphism located in the  HAMP gene promoter in TB patients and the control group and its relationship with serum iron, ferritin, and hepcidin. In the present work, the correlation of   582A   G variants to TB disease was highly signifi cant (Table II) and no studies have been reported previously. Our results on the associa-tion of   582A   G polymorphism to serum iron and hepcidin are in agreement with the study by Andreani et  al. [17] and in contrast with work by Bruno et  al. that did not fi nd any association [19]. We report a reverse correlation between hepcidin and iron in the way that high hepcidin decreases the iron level (Table IV), as has been reported in previ-ous studies [7,23,24]. The   582A   G variants dem-onstrated an impact on the level of hepcidin expression and its subsequent increase in serum iron in TB patients. These observations suggest that   582A   G polymorphism may be associated with changes in the  HAMP promoter function. This  HAMP promoter substitution, therefore, might rep-resent a risk factor for TB patients by promoting iron overload in them.  Figure 1. Electrophoresis pattern of PCR for detection of   582A   G polymorphism.  Table II. The genotypes and alleles distribution of   582A   G polymorphism in case and control groups. CharacteristicTB group,  n (%)Control group  n (%)Odds ratio (95% CI)  p valueGenotypesAA 21 (20) 66 (63.5) 1GA 72 (68.6) 32 (30.8) 7 (3.7 – 13.5)    0.0001GG 12 (11.4) 6 (5.7) 6.1 (2.1 – 19.8) 0.0009A/G   G/G 84 (80) 38 (36.5) 6.9 (3.7 – 12.9)    0.0001AllelesA 114 (54.3) 164 (78.9) 1G 96 (45.7) 44 (21.1) 3.1 (2 – 4.8)    0.0001 CI, confi dence interval; TB, tuberculosis.  Table III. Levels of serum iron, ferritin, and hepcidin according to  HAMP   582A   G genotype in patients with tuberculosis (TB).   582A   G genotypesCharacteristic A/A A/G   G/G  p value Normal range n (%) 21 (20) 84 (80)Serum iron, mean   SD ( μ g/dl) 31.6    17.7 53.5    16.2 0.003 45 – 160 males; 40 – 150 femalesSerum ferritin, mean   SD (ng/ml) 31.9    28.7 39.6    35.5 0.045 10 – 295 males; 25 – 325 femalesSerum hepcidin (ng/ml), geometric mean (95% range) 57.5 (21.9 – 88.7) 35.2 (17.4 – 53.1) 0.030 13.3 – 54.4 adults696 M. Javaheri-Kermani et al.  Conclusion  Due to the impact of hepcidin on iron level, differ-ences in the level of hepcidin expression may par-tially relate to the phenotypic variants in iron hemostasis among TB patients. In the present study, a high association was found between serum hepci-din level and the  HAMP   582A   G variants in TB patients. These observations indicate a hypothetical role of this polymorphism in iron metabolism. This assay can be useful as part of the diagnostic and prognostic evaluation in TB disease. Hepcidin could perhaps be an option for the treatment of TB.  Declaration of interest:  The authors report no confl icts of interest. The authors alone are respon-sible for the content and writing of the paper.  This study was prepared from Mina Javaheri-Kermani ’ s MSc thesis and supported by grants from Golestan University of Medical Sciences and Mazandaran University of Medical Sciences.  References  Eurosurveillance editorial team.  WHO publishes Global [1] tuberculosis report  2013 .  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Impact of hepcidin antimicrobial peptide on iron overload in tuberculosis patients

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