Possible role of PGD2in malaria infections

Pimwan Thongdee， Jiraporn Kuesap， Raewadee Wisedpanichkij， Kesara Na-Bangchang，4✉

1Graduate Program in Bioclinical Sciences， Chulabhorn International College of Medicine， Thammasat University， Pathumthani， Thailand

2Graduate Program in Biomedical Sciences， Faculty of Allied Health Sciences， Thammasat University， Pathumthani， Thailand

3Department of Hematology， Rama College of Medicine， Mahidol University， Bangkok， Thailand

4Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma， Thammasat University， Pathumthani， Thailand

Possible role of PGD2in malaria infections

Pimwan Thongdee1， Jiraporn Kuesap2， Raewadee Wisedpanichkij3， Kesara Na-Bangchang1，4✉

1Graduate Program in Bioclinical Sciences， Chulabhorn International College of Medicine， Thammasat University， Pathumthani， Thailand

2Graduate Program in Biomedical Sciences， Faculty of Allied Health Sciences， Thammasat University， Pathumthani， Thailand

3Department of Hematology， Rama College of Medicine， Mahidol University， Bangkok， Thailand

4Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma， Thammasat University， Pathumthani， Thailand

ARTICLE INFO

Article history:

Received 15 May 2016

Received in revised form 16 June 2016

Accepted 1 July 2016

Available online 20 September 2016

Malaria

Plasmodium vivax

Plasmodium falciparum

Prostaglandin D2（PGD2）

Malaria severity

Objective: To preliminarily investigate the possible role of prostaglandin D2（PGD2） in malaria infections. Methods: Blood and urinary samples （n=120 each） were collected from Thai patients with Plasmodium falciparum （P. falciparum） with moderate （n=26） and high （n=4）parasitemia， patients with Plasmodium vivax （P. vivax） （n=30）， patients with fever associated with other infections （n=30）， and healthy subjects （n=30）. PGD2concentrations in plasma and urinary samples of healthy subjects， patients with fever associated with other infections and patients with malaria were determined using Prostaglandin D2-MOX express EIA kit （Cayman Chemical， USA）. Results: The possible association between PGD2and malaria infections is clearly demonstrated with PGD2concentration in urine. The urinary PGD2concentrations were relatively high （about 5-fold） in patients with P. falciparum with moderate parasitemia and P. vivax infections compared with other groups. Furthermore， the concentration in patients with P. falciparum with moderate parasitemia and P. vivax infection were signif i cantly higher than that in healthy subjects and patients with fever associated with other infections. Conclusions: Urinary PGD2concentrations may of f er a more dependable and useful tool for predicting malaria severity. Conf i rmation is this preliminary fi nding is required with a larger sample size.

1. Introduction

Globally， an estimated 3.2 billion people in 97 countries and territories are at risk of being infected with malaria and developing disease and 1.2 billion are at high risk. According to the latest estimates， 198 million cases of malaria occurred globally in 2013 and the disease led to 584 000 deaths， representing a decrease in malaria case incidence and mortality rates of 30% and 47% since 2000， respectively［1］. Among all human malaria species，Plasmodium falciparum （P. falciparum） is the most severe form with regard to morbidity and mortality. Several factors associated with pathogenesis and severity of severe P. falciparum have been reported， but major factors involve the production of cytokines（IL-4， IL-12） and tumor necrosis factor （TNF）［2，3］. Recently，the hypothetical role of hemeoxygenase-1 （HO-1） enzyme in pathogenesis of severe and cerebral malaria has been proposed as one of the important factors that may be linked with susceptibility and severity of malaria infections［4］. HO-1 is the enzyme involved in heme breakdown process to release iron， carbon monoxide， and biliverdin/bilirubin. This process therefore inf l uences iron supply that support the growth of P. falciparum［5］. The expression of HO-1 is induced by several substances particularly prostaglandin D2（PGD2）. PGD2is the most important prostanoid produced in the brain that regulates sleep and pain responses［5］. The production of PGD2is induced through transcriptional activation of cyclooxygenase-2， as well as heme degradation［6］. It is thoughtthat intra-erythrocytic P. falciparum parasites release PfPGD2which may inf l uence heme catabolism in the host cells near the parasite sequestration sites. The sequestered parasitized erythrocytes then generate hemodynamic stress， which in turn， increase the production of PfPGD2through induction of the expression of lipocalin-type prostaglandin D2synthase in vascular endothelial cells as reported in the case of fl uid shear stress［7］. The aim of the present study was to preliminarily investigate the possible role of PGD2in malaria infections through measuring its concentrations in blood and urinary samples collected from Thai patients with P. falciparum， Plasmodium vivax （P. vivax）， patients with fever， and healthy subjects of both genders and all age groups.

2. Material and methods

2.1. Study areas and sample collection

The study was conducted at Mae Sot General Hospital， Mae Sot， Tak Province， Thailand. Approval of the study protocol was obtained from the Ethics Committees of Ministry of Public Health of Thailand. A total of 120 blood and urine samples were collected（before treatment） from Thai patients with P. vivax （n=30）， patients with P. falciparum ［n=26 and 4 for moderate （1 000-50 000 asexual parasite/μL）， and high parasitemia （＞50 000 asexual parasite/μL），respectively）］， patients with fever associated with other infections（n=30）， and healthy subjects （n=30）. Written informed consents for study participation were obtained from all participants ［89 males and 31 females， aged （12-90） years］ before study.

Blood sample （3 mL） was collected from each participant and serum and plasma （with EDTA anticoagulant and 10 μM indomethacin） samples were prepared through centrifugation at 1 500×g for 15 min （4 ℃）. Random mid-stream urine sample （2 mL）was collected and immediately stored at -80 ℃ without pretreatment with any preservative.

2.2. Sample extraction

Plasma sample was diluted with cold acetone at the ratio of 1:1（v: v） and incubated on ice for 5 min. The precipitated protein was removed through centrifugation at 8 000×g for 10 min and stored at -20 ℃ until analysis. Before analysis， the sample was evaporated to dryness using Centri-vap cold trap and the dried sample was resuspended in 100 μL of EIA buf f er. Methoximation was performed using Prostaglandin D2-MOX express EIA kit according to the procedure provided by the manufacturer. Concentrations of bilirubin （direct and total） in serum samples were determined immediately after sample collection by diazonium salt 3， 5 dichlorophenyldiazonium tetrafluoroborate （DPD） method. Creatinine concentrations in urine samples were determined using Jaf f e’s reaction method.

2.3. Determination of PGD2concentrations

PGD2concentrations in plasma and urine samples were determined using Prostaglandin D2-MOX express EIA kit （Cayman Chemical，USA）. Briefly， 50 μL of plasma （1: 10 dilution） or urine （1: 5 dilution） was added in a 96-well plate coated with goat anti-rabbit IgG antibodies. The tracer （50 μL） and the PGD2specif i c antibody（50 μL） were added to each well. The plate was incubated overnight at 4 ℃ and washed fi ve times with 10 mM phosphate buf f er （pH 7.4） containing Tween 20 （0.05%） pH 7.4. Two-hundred μL of Ellman’s reagent ［69 mM acetylthiocholine and 54 mM 5， 50-dithiobis （2-nitrobenzoic acid） in 10 mM phosphate buf f er pH 7.4］ was added to each well and the plate was incubated in a dark room at 25 ℃ for （60-90） min. Concentration of the reaction product （yellow solution） was spectroscopically measured using a microplate reader at the wavelength of 410 nm. A standard curve was developed using computer spreadsheet of Cayman Chemical Company and concentrations of PGD2in plasma and urine samples relative to those standards were determined.

2.4. Statistical analysis

Dif f erence in PGD2concentrations in samples of all groups were determined using Kruskal-Wallis test followed by Mann-Whitney U test for data not conforming to normal distribution. Statistical signif i cance level was set at α = 0.05 for all tests.

3. Results

3.1. Association between plasma PGD2concentrations and malaria infections

Median （range） values of plasma PGD2concentrations in samples collected from patients with P. vivax， patients with P. falciparum（moderate and high parasitemia）， patients with fever associated with other infections， and healthy subjects are summarized in Table 1. The concentration was highest （about 3-fold） in patients with fever associated with other infections compared with other groups. The concentration in P. falciparum infection with moderate parasitemia was significantly higher than healthy subjects （P ＜ 0.000 1）， but signif i cantly lower than P. vivax infection （P ＜ 0.000 1） and patients with fever-associated with other infections （P ＜ 0.000 1）. For P. vivax infection， the concentration was significantly higher than patients with P. falciparum with moderate parasitemia （P ＜ 0.000 1） and healthy subjects （P ＜ 0.000 1）， but signif i cantly lower thanpatients with fever-associated with other infections （P ＜ 0.05）

Table 1Median （range） plasma concentrations of PGD2， serum total bilirubin concentration， serum direct bilirubin concentration， urinary PGD2concentration and urinary creatinine concentration in 4 groups.

3.2. Association between serum bilirubin concentrations and malaria infections

Median （range） values of serum concentrations of bilirubin （direct and total） in samples collected from all groups are summarized in Table 1. The concentrations in patients with P. falciparum with moderate parasitemia and P. vivax infections were significantly higher than healthy subjects （P ＜ 0.05 and P ＜ 0.05， respectively）. The increased bilirubin level did not interfere the measurement of serum PGD2level.

The median （range） direct bilirubin concentrations in patients with P. falciparum with moderate parasitemia and P. vivax infections were signif i cantly higher than healthy subjects （P ＜ 0.05 and P ＜ 0.05，respectively）. In addition， the concentration in patients with feverassociated with other infections was signif i cantly higher than healthy subjects （P ＜ 0.05）.

3.3. Association between urinary PGD2concentrations and malaria infections

Median （range） values of urinary PGD2concentrations in plasma samples collected from all groups are summarized in Table 1. The median urinary PGD2concentrations were relatively high （about 5-fold） in patients with P. falciparum with moderate parasitemia and P. vivax infections compared with other groups. The concentration in patients with P. falciparum with moderate parasitemia was significantly higher than that in healthy subjects （P ＜ 0.05） and patients with fever associated with other infections （P ＜ 0.05）. For P. vivax infection， the concentration was signif i cantly higher than that in healthy subjects （P ＜ 0.05） and patients with fever associated with other infections （P ＜ 0.05）.

3.4. Association between urinary creatinine concentrations in urine samples and malaria infections

Median （range） values of urinary creatinine concentrations in samples collected from all groups are summarized in Table 1. The concentration in patients with P. falciparum with moderate parasitemia was signif i cantly higher than healthy subjects （P ＜ 0.05）and patients with fever associated with other infections （P ＜ 0.05）. For P. vivax infection， the concentration was significantly higher than healthy subjects （P ＜ 0.05） and patients with fever associated with other infections （P ＜ 0.05）.

4. Discussion

PGD2is markedly produced in the human brain to control sleep and pain responses. PGD2is also synthesized in mast cells and leukocytes by a cellular， myeloid-type， glutathione-dependent PGD2synthase［7］. PfPGD2is a potential factor derived from intraerythrocyte falciparum parasites. In a previous study in human astrocyte cell line （CCF-STTG1）， PGD2increased the expression of HO-1 mRNA in a dose- and time-dependent manner. Therefore，PGD2might be involved in the pathogenesis of cerebral malaria through the induction of HO-1 expression in malaria patients［5］. In another study in human retinal pigment epithelial cells （ARPE-19，D407）， PGD2was shown to stimulate the expression of HO-1 mRNA and protein through binding to prostaglandin-D2receptor （DP2），linking the PGD2-DP2with heme homeostasis［8］. Study in Gambian children with severe malaria demonstrated that （GT） （n） repeat polymorphism in the HMOX1 promoter inf l uenced the magnitude of HO-1 gene expression， while high HO-1 level was associated with severe disease［4］. The present study aimed to investigate the possible link between PGD2concentrations in plasma and urinary samples and malaria infections， through the induction of HO-1 enzyme. Plasma PGD2concentration was shown to be signif i cantly higherin patients with P. falciparum infection with moderate and high parasitemia compared with healthy subjects. It was noted however that， PGD2concentrations in patients with P. vivax infection and those with fever associated with other infections were respectively，about 2- and 4-fold of that in healthy subjects. The variability of the PGD2concentrations measured could be due to interference from other substances in plasma samples particularly bilirubin. Signif i cantly higher serum total and direct bilirubin concentrations were observed in samples collected from malaria patients compared with healthy subjects. Moreover， PGD2-EIA anti-serum derived from human PGD2used in the EIA method might be non-specif i c to PfPGD2. This may suggest the dif f erence between human PGD2and PfPGD2［10］.

The possible association between PGD2and malaria infections is clearly demonstrated with PGD2concentration in urine. The urinary PGD2concentrations were relatively high （about 5-fold） in patients with P. falciparum with moderate parasitemia and P. vivax infections compared with other groups. Furthermore， the concentration in patients with P. falciparum with moderate parasitemia and P. vivax infection were signif i cantly higher than that in healthy subjects and patients with fever associated with other infections. Urinary PGD2concentrations may of f er a more dependable and useful tool for predicting malaria severity. Plasma PGD2is not a suitable matrix due to rapid degradation of PGD2in the presence of plasma protein especially albumin， which complicates the analysis of PGD2［9］. For determination of PGD2in plasma as well as tissue homogenates，samples needs to be extracted immediately after collection to remove proteins and to stabilize PGD2. Indomethacin was immediately added to whole blood sample after collection to prevent ex vivo formation of eicosanoids which have the potential to interfere with the EIA assay. For urinary samples however， the addition of indomethacin is not required［7］. Further study in a larger sample size should be performed to conf i rm the possible association between PGD2levels and malaria infections including its predicting tool for malaria disease severity. One limitation of the study is that spot urine was used to measure the PGD2levels. The spot urine can be diluted or concentrated depending on patients’ urine volume and renal function.

Acknowledgments

The study was supported by The Commission on Higher Education，Ministry of Education of Thailand， The National Research University Project of Thailand （NRU）， Offi ce of Higher Education Commission，Thammasat University （Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma）， and The Royal Golden Jubilee PhD Programme， Thailand Research Fund -Thammasat University Joint Fund and Graduated Student Grant to P. Thongdee （No. PHD/0365/2552）.

Conflict of interest statement

We declare that we have no conf l ict of interest.

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10.1016/j.apjtm.2016.07.006

Pimwan Thongdee， Graduate Program in Bioclinical Sciences， Chulabhorn International College of Medicine， Thammasat University， Pathumthani， Thailand.

✉Corresponding author: Kesara Na-Bangchang， Graduate Program in Bioclinical Sciences，Chulabhorn International College of Medicine， Thammasat University， Pathumthani，Thailand.

E-mail: kesaratmu@yahoo.com

Tel: 662 5644440-79 （ext. 1803）

Fax: 662 5644398

Foundation project: This research was funded by Thailand Research Fund-Thammasat University Joint Fund and Graduated Student Grant to P. Thongdee （No. PHD/0365/2552）.