Β-defensin Research Articles

Prokaryotic expression of mouse interleukin 17A and its ability of stimulating RAW264.7 cell inflammatory factors expression

Objective To express and purify mouse interleukin 17A(mIL-17A) in E. coli and to analyze its ability of stimulating macrophage inflammatory factors expression. Methods The coding gene of mouse mIL-17A mature protein was amplified from mouse spleen cells by RT-PCR. PCR product was cloned into the prokaryotic expressing vector pET28a, and the resulting recombinant plasmid pET28a/mIL-17a was then transformed into the host E. coli strain BL21(DE3) for expression. The mIL-17A protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by the Ni-NTA affinity chromatography, and was further tested on the stimulation of cytokine and chemokine of RAW264.7 cells by ELISA and real-time quantity PCR in vitro. Results The mIL-17A with bioactivity was over-expressed and purified successfully, and the results of real-time PCR and ELISA showed that recombinant mIL-17A stimulated macrophage mRNA upregulation of IL-6, defensin β2 and Cxcl3 and secretion of defensin β2, Ccl3, Cxcl3,IFN-γ, IL-6 and IL-4. Interestingly, these effects could be blocked by the addition of anti-IL-17A neutralizing antibody partly. After treatment with mIL-17, 74. 87-fold of defensin β2 mRNA expression was increased comparing with that of untreated cells( P ＜0.01 ), while blocking with anti-IL-17A antibody the increase was only 5.4-fold(P ＜ 0.01 ). Conclusion The recombinant mIL-17A has a strong stimulation on secretion of cytokine and chemokine of macrophage, that maybe result to the enhancement of anti-infection ability of macrophage. Key words: Recombinant interleukin 17A; Macrophage inflammatory protein; Anti-infection immunity; Beta-defensin

Legionella pneumophila induces human beta Defensin-3 in pulmonary cells

BackgroundLegionella pneumophila is an important causative agent of severe pneumonia in humans. Human alveolar epithelium and macrophages are effective barriers for inhaled microorganisms and actively participate in the initiation of innate host defense. The beta defensin-3 (hBD-3), an antimicrobial peptide is an important component of the innate immune response of the human lung. Therefore we hypothesize that hBD-3 might be important for immune defense towards L. pneumophila.MethodsWe investigated the effects of L. pneumophila and different TLR agonists on pulmonary cells in regard to hBD-3 expression by ELISA. Furthermore, siRNA-mediated inhibition of TLRs as well as chemical inhibition of potential downstream signaling molecules was used for functional analysis.ResultsL. pneumophila induced release of hBD-3 in pulmonary epithelium and alveolar macrophages. A similar response was observed when epithelial cells were treated with different TLR agonists. Inhibition of TLR2, TLR5, and TLR9 expression led to a decreased hBD-3 expression. Furthermore expression of hBD-3 was mediated through a JNK dependent activation of AP-1 (c-Jun) but appeared to be independent of NF-κB. Additionally, we demonstrate that hBD-3 elicited a strong antimicrobial effect on L. pneumophila replication.ConclusionsTaken together, human pulmonary cells produce hBD-3 upon L. pneumophila infection via a TLR-JNK-AP-1-dependent pathway which may contribute to an efficient innate immune defense.

Beta defensin‐1 gene (DEFB1) polymorphisms are not associated with atopic dermatitis in children and adolescents from northeast Brazil (Recife, Pernambuco)

Atopic dermatitis (AD) is a common inflammatory skin disease resulting from the interplay between environmental, immunological and genetic factors. In our study, we investigated the role of three single nucleotide polymorphisms (SNPs) at 5'-UTR of DEFB1 gene, encoding for the human beta defensin-1, on the susceptibility to develop AD in a group of Brazilian children and adolescents. Three SNPs, -20 G/A (rs11362), -44 C/G (rs1800972), and -52 G/A (rs1799946) at 5'-UTR of DEFB1 gene were genotyped in two groups of children and adolescents, one affected by AD (96 subjects), the other healthy (191 individuals), from northeast Brazil. -44 C/G frequencies were comparable between the two groups. The -20 GG genotype was more frequent in AD subjects than in healthy controls; the -52 GG, conversely, was more frequent in healthy controls than in AD. However, both these differences did not reach statistical significance. Also, association between SNPs and AD severity has been shown. The analysis of DEFB1 haplotypes did not highlight any association of the three SNPs with AD development or disease severity. Our results seem to exclude a role for the -44 C/G DEFB1 SNPs on the pathogenesis and severity of AD, while for the -20 C/G and -52 G/A, even if not statistically significant, we evidenced a slight trend for susceptibility (-20 GG) and protection (-52 GG) for the development of AD. However, as controversial findings have been reported in the literature, the role of DEFB1 in the development of AD and in the severity of the phenotype deserves further investigation.

Analysis of DEFB1 regulatory SNPs in cystic fibrosis patients from North‐Eastern Italy

Cystic fibrosis (CF) transmembrane regulator protein (CFTR) gene is undoubtedly the main genetic factor involved in the modulation of CF phenotype. However, other factors such as human defensins and the genes encoding for these antimicrobial peptides have been hypothesized as possible modifiers influencing airways infection in CF patients, but their role in the pathogenesis of lung disease is still debated. Since DEFB1 gene encoding for human beta-defensin 1 displays features such as antimicrobial or chemotactic activity playing a role in inflammation, it has been considered as a possible candidate CF modifier gene. We analysed three single nucleotide polymorphisms (SNPs) in the 5'-untranslated region of the DEFB1 gene (namely g-52G>A, g-44C>G and g-20G>A) in a group of 62 CF patients from North Eastern Italy, and in 130 healthy controls, with the aim of verifying the possible association of these functional SNPs with the pulmonary phenotype of CF patients. DEFB1 SNPs have been genotyped by using Taqman allele-specific fluorescent probes and a real-time PCR platform. No significant differences were found for allele, genotype and haplotype frequencies of DEFB1 g-52G>A, g-44C>G and g-20G>A SNPs in CF patients stratified for Pseudomonas aeruginosa infection, as well as in patients with a severe and mild clinical phenotype or in patients stratified for CFTR genotypes. DEFB1 allele, genotype and haplotype frequencies of CF patients globally considered were similar to those of healthy controls. Our findings are discordant with respect to another recent study performed on CF patients coming from Southern Italy, probably due to different ethnicity of the patients.

Dissection of Host Cell Signal Transduction during Acinetobacter baumannii – Triggered Inflammatory Response

Infected airway epithelial cells up-regulate the expression of chemokines, chiefly IL-8, and antimicrobial molecules including β-defensins (BD). Acinetobacter baumannii is a cause of hospital-acquired pneumonia. We examined whether A. baumannii induced the expressions of IL-8 and BD2 by airway epithelial cells and the receptors implicated in bacterial detection. A549 and human primary airway cells released IL-8 upon infection. A. baumannii-infected cells also increased the expression of BD2 which killed A. baummannii strains. IL-8 induction was via NF-κB and mitogen-activated kinases p38 and p44/42-dependent pathways. A. baumannii engaged Toll-like receptor (TLR) 2 and TLR4 pathways and A549 cells could use soluble CD14 as TLRs co-receptor. A. baumannii lipopolysaccharide stimulated IL-8 release by A549 cells and sCD14 facilitated the recognition of the lipopolysaccharide. Mass spectrometry analysis revealed that A. baumannii lipid A structure matches those with endotoxic potential. These results demonstrate that airway epithelial cells produce mediators important for A. baumannii clearance.

The Antimicrobial Peptide DEFB1 Is Associated with Caries

Genetics is an important component in the determination of individual susceptibility to caries and periodontal diseases. Since beta defensin 1 (DEFB1) localizes in the oral cavity, we tested if variation in DEFB1 is associated with caries and periodontitis. We analyzed 3 single-nucleotide polymorphisms in DEFB1 in DNA samples from unrelated individuals. Carrying a copy of the variant allele of the DEFB1 marker rs11362 (G-20A) increased the DMFT and DMFS scores more than five-fold. Also, carrying a copy of the variant allele of the DEFB1 marker rs179946 (G-52A) correlated with low DMFT scores. We found a high-caries-experience haplotype (GCA), which increased DMFT scores two-fold, and a low- caries-experience haplotype (ACG), which decreased DMFT scores two-fold, in the DEFB1 promoter. No association between DEFB1 genetic markers and periodontal disease was found. Our results suggest that functional polymorphisms of DEFB1 are potential markers for caries.

Overexpression of Act1 drives beta‐defensin expression

Overexpression of Act1 (CIKS) drives the expression of the human beta defensin gene, DEFB4, in a human bronchial epithelial cell line (HBE1). Act1 is an adaptor protein that is known to interact with I‐kappa‐kinases, as well as the interleukin‐17 receptor chain IL17RA, and is a potent activator of NFκB. We have previously shown that induction of DEFB4 by interleukin‐17 is largely NFκB dependent. Using DEFB4 promoter‐reporter constructs, we surprisingly show that induction of DEFB4 by Act1 overexpression is NFκB independent. Furthermore, we show that induction depends primarily on a region of the DEFB4 promoter that is located between −2.2 kb and −1 kb upstream of the transcriptional start site.This research was supported in part by grants from the NIH (HL077902, HL077315, ES00628)

Role of human beta defensin 3 during type I interferon mediated antiviral response against vesicular stomatitis virus

Type I interferons (IFN), IFN-α/β constitute a critical component of innate immunity against viruses. IFN-α/β activates JAK/STAT pathway resulting in expression of various IFN- stimulated antiviral proteins. While studying IFN-α/β signaling during virus infection, we identified human beta defensin-3 (HBD3) as an antiviral factor induced during IFN- α−mediated innate antiviral response in human lung epithelial cells. We showed that HBD3 is induced by IFN-α and purified HBD3 significantly inhibited vesicular stomatitis virus (VSV) infection. Further studies revealed that HBD3 confers its antiviral activity by blocking VSV cellular entry. An essential role of HBD3 during innate antiviral response was evident from loss of antiviral activity of IFN-α following HBD3 silencing by siRNA. Thus, we have identified HBD3 as an

Administration of nonviral gene vector encoding rat β‐defensin‐2 ameliorates chronic Pseudomonas aeruginosa lung infection in rats

Beta-defensin-2 (BD-2) plays an important role in host defense against pathogenic microbe challenge by its direct antimicrobial activity and immunomodulatory functions. The present study aimed to determine whether genetic up-regulation of rat BD-2 (rBD-2) could ameliorate chronic Pseudomonas aeruginosa lung infection in rats. Plasmid-encoding rBD-2 was delivered to lungs in vivo using linear polyethylenimine at 48 h before challenging with seaweed alginate beads containing P. aeruginosa. Macroscopic and histopathological changes of the lungs, bacterial loads, inflammatory infiltration, and the levels of cytokines/chemokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, kertinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2)] were measured at 3 and 7 days post-infection (p.i.). The overexpression of rBD-2 resulted in a significant increase in animal survival rate (at 3 days p.i.), a significant decrease in bacterial loads in the lungs (at 3 and 7 days p.i.), and significantly milder lung pathology. In addition, the overexpression of rBD-2 led to increased infiltration of polymorphonuclear neutrophils (PMN), and elevated protein expression of cytokines/chemokines (IL-1beta, TNF-alpha, KC and MIP-2) at the early stage of infection (at 3 days p.i.), at the same time as being dramatically decreased at the later stage of infection (at 7 days p.i.). Genetic up-regulation of rBD-2 increased animal survival rate, and reduced bacterial loads in lungs after bacterial infection. The overexpression of rBD-2 also modulated the production of several cytokines/chemokines and increased PMN recruitment at the early stage of infection. Our findings indicate that the enhancement of BD-2 may be an efficacious intervention for chronic P. aeruginosa lung infection.

Anal cytology screening in HIV-positive men who have sex with men: whatʼs new and whatʼs now?

This review will discuss current and future anal cytology screening programs to detect anal dysplasia in HIV-positive men who have sex with men (MSM), in addition to other interventions aimed at early detection of anal cancer in this population. Evidence of progression from high-grade anal dysplasia to anal cancer has been recently demonstrated and strengthens the clinical imperative to diagnose and treat this lesion in at-risk populations. The use of adjunct molecular techniques that improve specificity of anal cytology screening may have the potential to rationalize current screening referral pathways and focus resources on those at highest risk of progressing to cancer. Candidate biomarkers that are currently being investigated include protein surrogates of cell cycle deregulation (such as p16ink4a and minichromosomal maintenance proteins), DNA damage biomarkers (53BP1) in addition to testing for human papillomavirus genotype, gamma tubulin and beta defensin levels. Anal cytology, although sensitive for the detection of any anal dysplastic abnormality, has poor specificity to detect high-grade anal dysplasia. As new molecular techniques are evolving, the most important immediate clinical intervention is to educate HIV-positive MSM in order to increase awareness of both risk and clinical symptoms suggestive of progression to anal cancer. In the absence of high-resolution anoscopic expertise, it is recommended that regular digital rectal examinations are performed as most early cancers are palpable even in the absence of clinical symptoms.

Toll-like Receptors and Antimicrobial Peptides Expressions of Psoriasis: Correlation with Serum Vitamin D Level

To evaluate the association of Toll-like receptors (TLRs), antimicrobial peptides (AMPs) and vitamin D receptors (VDRs) in psoriasis, lesional (PP) and perilesional skin (PN) from psoriasis, atopic dermatitis (AD) patients and healthy controls (NN) were studied by immunohistochemistry. Compared with PN, AD and NN skin, dysregulated expression of TLRs, AMPs and VDR was detected in PP skin. Noteworthy, our results showed altered correlation between TLR2 and VDR expression in PP and PN skin. Human beta defensin 2 (HBD2) and cathelicidin (LL-37) expressions in the PP skin were higher in serum vitamin D sufficient (VDS) groups than serum vitamin D deficient (VDD) groups. Negative correlation was found between TLR2 and VDR expression in the PP skin of VDD groups. However, positive correlation was noted in the PP skin of VDS groups. Based on the present results, therapies targeting the activity of TLRs, AMPs and vitamin D, including modulation of the TLR-VDR pathways, might provide new therapeutic approaches to the psoriasis and other inflammatory skin diseases.

Effect of polymorphisms of Crohn disease related NOD2 gene and human beta-defensin 2 on expression of human beta-defeusin 2

Objective To explore the effects of polymorphisms of Crohn＇s disease related NOD2 gene and human beta-defensin 2 （hBD-2） on transcription of hBD-2 gene and its mechanism. Methods I-IEK293T cells were transfected with hBD-2 gene and NOD2 eukaryotic expression plasmid, and were then stimulated with LPS, TNF-α or BAY 11-7082 （ antagonist of NF-κB） , respectively. Transcriptional activity of hBD-2 was detected afterwards. Results LPS could suppress transcription of hBD-2 （ P = 0. 020） , which was increased by TNF-α in a dose-dependent manner （P = 0. 004）. In the presence of LPS, there was significant difference in transcriptional activity of hBD-2 between wild-NOD2 transfected group and mutated NOD2 （P268S） transfected group （P = 0. 008 ）, but there was no significant difference between wild hBD-2 transfected group and mutated hBD-2 transfected group （ P =0. 053 ）. With the stimulation of TNF-α （5 ng/ml）, there was a significant difference between mutated hBD-2 transfected group and wild hBD-2 transfected group （ P = 0. 006 ）, but no significant difference between wild-NOD2 transfected and mutated NOD2 transfected group was defected （P =0. 064 ）. Pretreatment with BAY 11-7082 before TNF-α （5 ng/ml） significantly inhibited the transcriptional activity of hBD-2 （ P 〈 0.001 ）. Conclusion The polymorphism of NOD2 affects the innate expression of hBD-2, the polymorphism of site in hBD-2 promoter （-233） may lead to significant decline of the inducible expression of hBD-2, and NF-K B might be a key pathway that NOD2 protein mediates the expression of defensin. Key words: Human beta-defensin-2 ; CARD15/NOD2 ; Crohn disease ; Gene polymorphism ; Immunity, Natural

ASSOCIATION BETWEEN DEFB1 GENE HAPLOTYPE AND HERPES VIRUSES SEROPREVALENCE IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA

Recent studies investigated the role of an unusual immune response to infective agents in the etiology of acute lymphoblastic leukemia (ALL) in children. Human β-defensin-1 (hBD-1) is an anti-microbial peptide of the innate immune system, which exerts a killing role against pathogens. In the present study, three polymorphisms have been genotyped, namely, -52G/A, -44C/G and -20G/A, of DEFB1 gene, coding for hBD-1, in 40 ALL patients and 40 healthy children, and tested for an association between genetic variants of the protein and seroprevalence of antibodies for herpes viruses. The seroprevalence of cytomegalovirus (CMV), herpes simplex viruses (HSV) and Epstein-Barr virus (EBV) IgG antibodies in leukemic children was higher than that in controls (CMV: 61.5 vs. 27.3%, p = .008; HSV: 50 vs. 24.2%, p = .04; EBV: 61.3 vs. 46.2%, p = ns, respectively). Carriers of the GCA haplotype were found to have a significantly higher rate of immunization against CMV and HSV in ALL children compared to controls (CMV: 68 vs. 29%, p = .006; HSV: 56 vs. 26%, p = .04, respectively). No such observation was made when we analyzed the immunization against Epstein-Barr virus (EBV) by GCA haplotype in case and controls (58 vs. 40%, p = ns). These findings suggest that leukemic patients carrying untranslated variants of hBD-1 display a higher susceptibility to herpes viruses infections than controls.

Differential expression pattern of genes encoding for anti-microbial peptides in the fetal membranes of patients with spontaneous preterm labor and intact membranes and those with preterm prelabor rupture of the membranes

Objective. Increased amniotic fluid concentrations of anti-microbial peptides, components of the innate immune system, have been reported in patients with preterm labor (PTL) with intact membranes and intra-amniotic infection and/or inflammation (IAI), as well as in patients with preterm prelabor rupture of the membranes (PPROM). This study was designed to confirm these results using a targeted approach, detecting DEFA1, DEFB1, GNLY, and S100A9 gene expression in the choriamniotic membranes in pregnancies complicated with PTL and intact membranes or PPROM, with and without histologic chorioamnionitis.Study design. Human fetal membranes were obtained from patients in the following groups: (1) PTL with intact membranes (n = 15); (2) PTL with intact membranes with histologic chorioamnionitis (n = 12); (3) PPROM (n = 17); and (4) PPROM with histologic chorioamnionitis (n = 21). The mRNA expression of α-defensin-1, β-defensin-1, calgranulin B and granulysin in the fetal membranes was determined by qRT-PCR.Results. (1) The expression of α-defensin-1 mRNA in the fetal membranes was higher in patients with PTL and intact membranes with histologic chorioamnionitis, than those without chorioamnionitis (19.4-fold, p < 0.001); (2) Among patients with histologic chorioamnionitis, patients with PTL and intact membranes had a higher α-defensin-1 mRNA expression than those with PPROM (5.5-fold, p = 0.003); (3) Histologic chorioamnionitis was associated with a higher calgranulin B mRNA expression in the chorioamniotic membranes of patients with both PTL and intact membranes (7.9-fold, p = 0.03) and PPROM (7.6-fold, p < 0.0001); (4) The expression of calgranulin B mRNA in the fetal membranes was higher in patients with PTL and intact membranes without histologic chorioamnionitis than in those with PPROM without histologic chorioamnionitis (2.7-fold, p = 0.03); (5) There were no differences in the expression of β-defensin-1 and granulysin in the chorioamniotic membranes between the study groups even in the presence of histologic chorioamnioniotis.Conclusions. (1) Among patients with histologic chorioamnionitis, the mRNA expression of α-defensin-1 and calgranulin B in the fetal membranes of patients with PTL and intact membranes as well as that of calgranulin B in the fetal membranes of patients with PPROM is higher than in the membranes of those without histologic chorioamnionitis; (2) histologic chorioamnionitis is associated with differences in the pattern of α-defensin-1 mRNA expression in the fetal membranes in patients with PTL and intact membranes and those with PPROM.

Treatment of plaque psoriasis with the two-compound product calcipotriol/betamethasone dipropionate versus both monotherapies: An immunohistochemical study

The combination of calcipotriol (Cp) and topical corticosteroids increases efficacy and reduces side effects as compared to monotherapies. Previous studies suggest that such combinations may have an added value with respect to reduction of T-cell subsets. A two-compound product consisting of Cp and betamethasone dipropionate (BD) has become available for the treatment of psoriasis and is clinically superior to both monotherapies. No immunohistochemical data are available on the in vivo effects of the two-compound formulation versus monotherapy with respect to the three key processes in psoriasis pathogenesis: epidermal proliferation, epidermal differentiation and inflammation. Therefore, 18 patients were treated with the two-compound product, Cp monotherapy or BD monotherapy for 6 weeks and biopsies were taken before and after 4 and 6 weeks of treatment. These biopsies were stained immunohistochemically and analysed (semi)quantitatively. All treatments decreased the number of Ki-67+ cells and increased the keratin 15+ staining. A more pronounced reduction of epidermal and dermal T-cell markers and human beta defensin-2 was seen following combination treatment, compared with both monotherapies. In conclusion, the investigated markers of the skin immune system and epidermal proliferation indicated an added value of the two-compound product over both monotherapies.

Histone H4 Is a Major Component of the Antimicrobial Action of Human Sebocytes

Antimicrobial peptides, such as cathelicidin and beta defensins, directly kill microbes and have been detected in human sebaceous glands and cell lines. Despite the presence of several such peptides, the apparent abundance of these is insufficient for direct killing of most skin pathogens. In this study, we sought to determine which molecules provide the majority of antimicrobial peptide activity in human sebocytes. Acid-soluble protein extracts of SEB-1 sebocytes were separated by reverse-phase high-performance liquid chromatography and were assayed for their capacity to inhibit the growth of Staphylococcus aureus. Antimicrobial activity was isolated in a single major fraction and identified to be histone H4 by mass spectrometry and western blot analysis. The importance of histone H4 in the antimicrobial activity of sebocytes was confirmed by a specific neutralizing antibody and by direct demonstration that recombinant histone H4 had antimicrobial activity against S. aureus and Propionibacterium acnes. In addition, histone H4 enhanced the antimicrobial action of free fatty acids in human sebum. Taken together, these results indicate that the release of histone H4 by holocrine secretion from the sebaceous gland may play an important role in innate immunity.

HIV Type 1 Fails to Trigger Innate Immune Factor Synthesis in Differentiated Oral Epithelium

The oral mucosa is relatively resistant to human immunodeficiency virus type 1 (HIV-1) transmission. The mechanisms contributing to this resistance remain incompletely understood, but may include HIV-induced synthesis of innate immune factors. We used fully differentiated oral epithelium as a surrogate for the oral mucosa in vivo, exposed it to X4- and R5-tropic HIV-1 in culture, and quantified mRNA expression of six innate immune factors. Neither virus increased expression of human beta defensin 2 (hBD-2) mRNA over supernatants from uninfected lymphoblast controls. HIV-1 also failed to induce mRNA of four additional innate immunity-related genes. Similar results were obtained with oral monolayer epithelial cells. Interestingly, the X4-tropic virus inhibited mRNA expression of hBD-2, and of three of the other factors, at higher dosages in the differentiated oral epithelium but not the monolayers. The failure of HIV-1 to induce innate immune factors in the differentiated epithelium was not due to a lack of tissue penetration, as we detected fluorescence-tagged virions up to 30 mum deep from the apical surface. HIV-1 does not trigger de novo innate immune factor synthesis in oral epithelium, pointing to the role of a constitutive innate immunity for protection against HIV-1 in the oral cavity.

Increased expression of human beta-defensin 3 in mollusca contagiosum

The human beta defensins (hBDs)-2 and -3 are inducible antimicrobial peptides present in human skin. Besides an important role in fighting bacteria, they also have an antiviral function. Molluscum contagiosum (MC) is a cutaneous viral disease caused by the MC virus. Lesions show a tendency towards spontaneous regression, which might be caused by antiviral proteins such as defensins. We report a marked increase in hBD-3 immunoreactivity in MC lesions in contrast to hBD-2, which was only marginally increased. We suggest a role for the hBD-3 peptide in MC pathogenesis.

Evolutionary Trajectories of Primate Genes Involved in HIV Pathogenesis

The current availability of five complete genomes of different primate species allows the analysis of genetic divergence over the last 40 million years of evolution. We hypothesized that the interspecies differences observed in susceptibility to HIV-1 would be influenced by the long-range selective pressures on host genes associated with HIV-1 pathogenesis. We established a list of human genes (n = 140) proposed to be involved in HIV-1 biology and pathogenesis and a control set of 100 random genes. We retrieved the orthologous genes from the genome of humans and of four nonhuman primates (Pan troglodytes, Pongo pygmaeus abeli, Macaca mulatta, and Callithrix jacchus) and analyzed the nucleotide substitution patterns of this data set using codon-based maximum likelihood procedures. In addition, we evaluated whether the candidate genes have been targets of recent positive selection in humans by analyzing HapMap Phase 2 single-nucleotide polymorphisms genotyped in a region centered on each candidate gene. A total of 1,064 sequences were used for the analyses. Similar median K(A)/K(S) values were estimated for the set of genes involved in HIV-1 pathogenesis and for control genes, 0.19 and 0.15, respectively. However, genes of the innate immunity had median values of 0.37 (P value = 0.0001, compared with control genes), and genes of intrinsic cellular defense had K(A)/K(S) values around or greater than 1.0 (P value = 0.0002). Detailed assessment allowed the identification of residues under positive selection in 13 proteins: AKT1, APOBEC3G, APOBEC3H, CD4, DEFB1, GML, IL4, IL8RA, L-SIGN/CLEC4M, PTPRC/CD45, Tetherin/BST2, TLR7, and TRIM5alpha. A number of those residues are relevant for HIV-1 biology. The set of 140 genes involved in HIV-1 pathogenesis did not show a significant enrichment in signals of recent positive selection in humans (intraspecies selection). However, we identified within or near these genes 24 polymorphisms showing strong signatures of recent positive selection. Interestingly, the DEFB1 gene presented signatures of both interspecies positive selection in primates and intraspecies recent positive selection in humans. The systematic assessment of long-acting selective pressures on primate genomes is a useful tool to extend our understanding of genetic variation influencing contemporary susceptibility to HIV-1.

Association of a genetic polymorphism (-44 C/G SNP) in the human DEFB1 gene with expression and inducibility of multiple β-defensins in gingival keratinocytes

BackgroundHuman β-defensins (hBDs) are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP) in the 5' untranslated region of the hBD1 gene (DEFB1), denoted -44 (rs1800972), is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression.MethodsTransfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression.ResultsTransfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1β, TNFα, and interferon-γ (IFNγ). In contrast to constitutive expression levels, IFNγ-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression.ConclusionThe DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNγ inducibility of these β-defensins and is likely to be more protective in conditions that enhance IFNγ expression such as chronic periodontitis. These results suggest a complex relationship between genetics and defensin expression that may influence periodontal health and innate immune responses.