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. 2013 Oct 17;7(10):e2491.
doi: 10.1371/journal.pntd.0002491. eCollection 2013.

Human neutrophil peptide-1 (HNP-1): a new anti-leishmanial drug candidate

Affiliations

Human neutrophil peptide-1 (HNP-1): a new anti-leishmanial drug candidate

Sara Dabirian et al. PLoS Negl Trop Dis. .

Abstract

The toxicity of available drugs for treatment of leishmaniasis, coupled with emerging drug resistance, make it urgent to find new therapies. Antimicrobial peptides (AMPs) have a strong broad-spectrum antimicrobial activity with distinctive modes of action and are considered as promising therapeutic agents. The defensins, members of the large family of AMPs, are immunomodulatory molecules and important components of innate immune system. Human neutrophil peptide-1 (HNP-1), which is produced by neutrophils, is one of the most potent defensins. In this study, we described anti-parasitic activity of recombinant HNP-1 (rHNP-1) against Leishmania major promastigotes and amastigotes. Furthermore, we evaluated the immunomodulatory effect of rHNP-1 on parasite-infected neutrophils and how neutrophil apoptosis was affected. Our result showed that neutrophils isolated from healthy individuals were significantly delayed in the onset of apoptosis following rHNP-1 treatment. Moreover, there was a noteworthy increase in dying cells in rHNP-1- and/or CpG-treated neutrophils in comparison with untreated cells. There is a considerable increase in TNF-α production from rHNP-1-treated neutrophils and decreased level of TGF-β concentration, a response that should potentiate the immune system against parasite invasion. In addition, by using real-time polymerase chain reaction (real-time PCR), we showed that in vitro infectivity of Leishmania into neutrophils is significantly reduced following rHNP-1 treatment compared to untreated cells.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression and purification of rHNP-1.
A) Expression of rHNP-1 in pQE-30. SDS-PAGE analysis of the target peptide showed that HNP-1 was successfully expressed. B) Western blotting analysis of the expressed peptide. MW: 100-bp molecular weight marker. lane 1: before IPTG induction; lane 2 and 3: 4 hours after induction. C) HNP-1 purification on Ni-NTA column by FPLC. The identity and purity of each FPLC fraction were evaluated on 17.5% SDS-PAGE. As illustrated in this figure, the peptide elutes (lanes 1–3) had acceptable purity for further use. MW: 100-bp molecular weight marker; lane 1, 2 and 3: peptide elutes.
Figure 2
Figure 2. Anti-promastigote and anti-amastigote effects of rHNP-1.
A) Initial gating of stationary-phase promastigotes. B, C) Anti-promastigote effect of commercial and recombinant HNP-1. After 24 hours, untreated and treated parasites with different concentrations of rHNP-1 and commercial form were stained with PI followed by flow cytometric analysis. B) By increasing the concentration of rHNP-1 from 0 to 60 μg/ml (a, b, c, d and e related to 0, 10, 20, 40 and 60 μg/ml concentrations of peptide, respectively), the percentage of necrotic population has increased consequently. The figure showed the results of three independent experiments. C) The flow cytometric results of three independent experiments showed that the percentage of necrotic population after being treated with 10 and 40 μg/ml concentrations of rHNP-1 was significantly less than the percentage of necrotic population treated with similar concentrations commercial HNP-1. D) Anti-amastigote effect of rHNP-1. L. major-infected LM-1 macrophages were treated by rHNP-1 or unfolded HNP-1. Untreated, infected LM-1 cells were act as control. Extracted total genomic DNA from different groups was subject to real-time PCR assay in order to quantify DNApol and TBP. For each sample, PCR assay was performed in duplicate, and the experiment was performed three times. DNApol quantity was normalized to TBP quantity of each sample. Real-time PCR analysis revealed that the normalized DNApol quantity in rHNP-1-treated LM-1 cells was significantly less than untreated ones (p<0.05) and there is no significant difference between DNApol quantities in unfolded HNP-1-treated and untreated groups.
Figure 3
Figure 3. rHNP-1 increased neutrophil viability.
MTT assay of treated neutrophils with different concentrations of rHNP-1 (in the range of 0 (as control group) to 60 μg/ml) revealed that rHNP-1 increased neutrophil viability in comparison with control. Concentration of 20 μg/ml had the highest effect on neutrophil viability. Experiment was performed three times in triplicate. Data were represented as mean ± SD. * p<0.05 versus control.
Figure 4
Figure 4. Assessment of neutrophil apoptosis by flow cytometry.
Isolated neutrophils from healthy individuals (either in vitro Leishmania-infected or not) were treated by 20 μg/ml of rHNP-1 (in presence or absence of 20 μg/ml of class A CpG motif). Apoptosis was assessed by flow cytometry using FITC–annexin V and PI. 15000 events were counted per sample and the percentages of apoptotic, dead or viable cells were determined. This assay was performed on isolated PMNs from 10 healthy individuals (in ten separate experiments) in duplicate. Values are compared between treated (by rHNP-1 and/or CpG motif) and untreated neutrophils and each bar represents the mean ± SD of ten independent experiments. * 0.01<p<0.05, ** 0.001<p<0.01 and *** p<0.001 versus control of each group (uninfected or infected) A) After rHNP-1 treatment of uninfected neutrophils, the percentage of viable cells increased. In all other treated groups, the percentage of viable cells (and consequently total Annexin V+ cells) was the same as untreated groups. B) Apoptotic cells and dead cells had different percentages in total Annexin V+ population resulted in an apoptotic to dead ratio of more than 6 for uninfected and infected control groups to a ratio of less than 2.5 for all treated groups.
Figure 5
Figure 5. Effect of rHNP-1 and/or CpG motif on cytokine production from uninfected and infected neutrophils.
GM-CSF-treated neutrophils (either in vitro Leishmania-infected or not) were treated by rHNP-1 (in presence or absence of class A CpG motif). Culture supernatants were then collected and the levels of TNF-α (A), IL-8 (B) and TGF-β (C) were quantified by ELISA. This procedure was carried out separately on isolated neutrophils from 20 healthy individuals and the assay was performed in duplicate. Each bar represents mean ± SD. * 0.01<p<0.05, ** 0.001<p<0.01 and *** p<0.001 versus control of each group (uninfected or infected). ns: not significantly different from control A) Cell treatment by rHNP-1 or combination of rHNP-1 with CpG motif substantially increased TNF-α production from neutrophils (irrespective of being infected). B and C) rHNP-1 or its combination with CpG motif had different effect on IL-8 or TGF-β release from uninfected and infected cells; an increasing effect on uninfected neutrophils and decreasing effect on infected ones. CpG treatment of infected groups had a reduction effect on IL-8 and TGF-β productions.
Figure 6
Figure 6. rHNP-1 treatment of infected neutrophils caused a significant reduction in parasite infectivity, determined by real-time PCR.
L. major-infected neutrophils were treated by rHNP-1 or unfolded HNP-1. Untreated, infected neutrophils were as control. Extracted total genomic DNA from different groups was subject to real-time PCR assay in order to quantify DNApol. As a negative control, reactions without DNA template were also performed. For each sample, PCR was performed in duplicate, and the experiment was performed three times. The percentage of infectivity rate reduction was determined based on following equation: [1−(DNApol quantitytreated group/DNApol quantitycontrol group)×ばつ100. rHNP-1 caused infectivity rate reduction in 8 treated neutrophil samples (out of 10) in the range of 20% to 80% and a mean ± SD value of 45.79±17.90%. Significant difference was found in the mean percentages of infectivity rate reduction between rHNP-1-treated neutrophil samples and unfolded HNP-1-treated ones (46±18% versus 12±4%, p≈0.001), showing that antimicrobial activity of HNP-1 is relied on its correct folding.

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