Chronic infection during placental malaria is associated with up-regulation of cycloxygenase-2

Collection of placentas was done at delivery, at the maternity ward the Centre de Santé Roi Baudoin, Guediawaye, Dakar Sénégal. Details regarding enrolment and standard laboratory procedures have been reported elsewhere [29]. It involved women, more than 16 years, with an uncomplicated delivery of live newborns. For all women giving informed consent, an immuno-chromatography test (ICT, PfHRP2 MaKromed) was carried out on placental blood. A standard questionnaire was used to record information including: mother's age, gravidity, gestational age (determined by the date of the last menstrual period and morphometric measurement of the uterus during antenatal clinic visits) and history of malaria attacks. Babies and placentas were weighed immediately after birth. Gender and Apgar scores were recorded. LBW was defined as < 2,500 g, and prematurity as gestational age < 37 weeks. Women with haemoglobin (Hb) less than 11 g/dl were considered anaemic.

The HIV infection status of the participants was not established at the time of the study however, HIV sero-prevalence in Senegalese population was low (0.8% of parturients in Dakar) [30]. All ICT-positive women received chloroquine after delivery. Anaemic women received iron and folic acid supplements.

After collection, whole placentas were refrigerated until assessment (less than two hours after delivery). For each placenta, four biopsies were sampled [31], rinsed in phosphate buffer saline (PBS), rapidly frozen in liquid nitrogen and stored at -80°C until RNA extraction. An additional four biopsies were sampled for histology and immunohistochemistry studies.

Immediately after collection, samples for histology and immunohistochemistry were fixed in 4% buffered paraformaldehyde and processed as previously described [29]. 5 μm cryosections were stained either with haematoxylin-eosin (H&E) or with May-Grünewald-Giemsa (MGG). Quantification of parasites was done in placenta for 100 fields (magnification 1000×) of MGG stained sections and in blood by examination of Giemsa-stained thick smears. Haemozoin (HZ) was identified on sections as brown granular material with high refringence on polarization microscope, which differentiates then from formalin crystals. Contain in HZ was qualitatively assessed on sections as: no HZ, low HZ (1-2 crystals/field at ×1000) or high HZ (> 2/field).

Acute infections (AI) were defined by the presence of parasites on sections without malaria pigment, chronic infections (CI) by presence of parasites and pigment, and past infections (PI) by the presence of pigment alone. PfHRP2+ placentas with no evidence of parasites or pigment were classified as "no-parasite" (NP). For the control group all PfHRP2 rapid diagnosis test, parasites and pigment detection were negative in placenta and in the venous blood.

For immunochemistry, after blocking endogenous peroxidases and non-specific binding, tissue sections were incubated for 1 h at room temperature with primary antibodies or control isotypes and revealed using LSAB detection kit (DakoCytomation). Antibodies used were CD68 (PG-M1), and HLA-DR (TAL.1B5) from DakoCytomation mouse antibodies against. The number of cells in the intervillous space was counted over 100 fields (magnitude ×400).

Frozen biopsies were ground into powder in liquid nitrogen before homogenization in 4 M guanidium buffer. RNAs were extracted using standard phenol-chloroform procedure, precipitated with isopropanol and washed before quantification. Integrity of total RNA was assessed on an ethidium bromide (EtBr) stained denaturing agarose gel. For reverse transcription 2 μg of RNA was denatured at 65°C for 10 min, cooled on ice-water, and reverse-transcribed for 60 min at 43°C with 40 U/μl RNase inhibitor and oligo-dT. Amplification was performed in an ABI Prism 7000 Sequence Detection System using Taqman Universal PCR Master Mix (PE Biosystems), containing 300 nM of primers and 200 nM of fluorogenic probes. Each measurement was carried out in duplicate and amplification of Peptidyl Propyl Isomerase A (PPIA, also called Cyclophylin A) was conducted on the same plate. Others housekeeping genes such as beta-2-microglobulin and beta-actin were run together with PPIA, but were not selected for standardization as less constant between infected and uninfected placentas. Probes and primers (Table 1) were designed using Primer Express software (Applied Biosystems). Cycling conditions started at 50°C for 2 minutes followed by 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The specificity of PCR amplifications was confirmed for each set of primers by sequencing of the PCR product and primers specificity was confirmed by single peaks demonstrated by dissociation curves after amplification of cDNA. The amplification efficiency was assessed for all the genes by amplifying serial dilutions of cDNA, and calculation of the slope (delta Ct)/(log cDNA dilution) which must be less than 0.1. Quantification of gene expression was done according to Livak et al[32] using for each gene the mean Ct-values of the duplicate. For each sample the results were adjusted according to the level of PPIA in the same sample. The PfHRP2-negative control group was used to normalize the results. For each gene, the relative quantity (RQ) of mRNA was expressed, in arbitrary units, as a fold increase in comparison with the control group.

Differences between groups were compared using Mann-Whitney U test (MW) for two groups or with Kruskal-Wallis test (KW) and Median test for more than two. Correlations were calculated using a Spearman R test (SP). Alpha risk was set at 0.05.

During this study, 692 pregnant women were examined among whom 71 had a Plasmodium falciparum histidine rich protein positive-RDT (PfHRP2+) (10.3%). For these PfHRP2+ women, 25% of the babies had LBW and 67% of the women were anaemic [29]. Compared to the control women, chronic-active infections were associated with lower birth weight (p < 0.008 and p < 0.0002 respectively) as well as CI and PI correlated with lower haemoglobin levels (p < 0.05 for both). No significant change was found for birth weight and haemoglobin, according to parity may be due to the low sample size of the study. For the purpose of this study, 54 placental samples were randomly selected from the different histological groups after classification (table 2). Among the selected PfHRP2+ women, 13 were primiparous, 13 secundiparous, and 14 multiparous.

COX-2 mRNA increased during PM especially during late stage of the infection: 9.0, 5.4, 3.8 and 3-time higher in placentas with CI, AI, PI and NP, respectively, than in the control group (p < 0.01 MW for CI and AI). The expression of COX-1 remained low in all the placentas without significant differences between PfHRP2- (Ctrl) and PfHRP2+ (CI, AI, PI & NP) groups, but trends to increase during past infection (Figure 1A).

Because IL-10 has been shown to exert direct regulatory effects on prostaglandin production, we also measured IL-10 mRNA in our placental samples. The levels of IL-10 transcripts were higher in PfHRP2+-placentas than in controls (MW p = 0.004, Figure 1A) and correlated with COX-2 expression (n = 54, SP p = 0.006 R = 0.36; and p = 0.008 R = 0.36). However none of these markers correlated with the parasitaemia. In the opposite way 15-LOX was lower in PfHRP2+-placentas than in controls (MW p = 0.0002) (Figure 1A), but correlates with parasitaemia in the placenta (SP R = 0.416, p = 0.0076) and with COX-2 mRNA expression (SP p = 0.018, R = 0.37).

The levels of COX-2 mRNA but not of COX-1, 15-LOX, and IL-10, trend to decrease when parity increased (Figure 1B), but there was no significant difference for any of these genes according to duration of the pregnancy or with the presence of fever at delivery.

Birth weight was significantly lower in primiparous than in secundiparous or multiparous (MW p < 0.05 for both). It was also lower during AI and CI than in control group. However, women who delivered babies with normal birth weight had higher level of COX-2 transcripts compared to others (MW p < 0.02; Figure 2B). In the opposite way COX-1 and 15-LOX transcripts trend to increase in association with LBW (respectively 2 and 1 fold increase than for the normal birth weight group, NS Figure 2B).

Haemoglobin was inversely related to the level of COX-1, COX-2 and IL-10 but increased with 15-LOX (SP R = -0.58, -0.57, -0.51, 0.48 respectively, p < 0.001 for each) (Figure 2A). Haemoglobin tends to decrease with the amount of HZ in the placenta (Figure 3A, middle panel; NS). Anaemia was thus associated with an increase of COX-2 and with PI (MW p < 0.001, with control group).

High level of pigment in the placenta was associated with significantly lower birth weight (KW p = 0.013, n = 44, Figure 3A, left panel), and with lower haemoglobin level in the mother (KW p = 0.018, Figure 3A, middle panel). However lower pigment density associated with a higher level of COX-2 and IL-10 transcripts (Figure 3d), without significant change for COX-1 (MW, p < 0.001 for both).

As detected by immunohistochemistry (Figure 3c), macrophages (CD68+) and activated HLADR+ presenting cells increased in PfHRP2+-placenta (p < 0.001 for both, DNS), mainly during late infection (CI or PI versus Ctrl: MW p < 0.001). Primiparous and secondiparous women exhibited higher levels of CD68+ cells when compared to multiparous (p < 0.05) while CI had significantly higher levels of CD68+ cells compared to PfHRP2- or control (p = 0.002). The density of macrophages (CD68) increased in the placenta when the amount of pigment was low (median test p < 0.0001). The count of macrophage was significantly correlated with COX-2 (SP p = 0.014, R = 0.331), IL-10 (SP p = 0.014, R = 0.332) and 15-LOX mRNA (SP p = 0.0003, R = 0.539). In the placental tissue sections COX-1 protein was detected by immunolabelling in trophoblasts, mononuclear interstitial cells and, most often, in foetal endothelial cells. COX-2 and 15-LOX were mainly detected in macrophages, endothelial cells and trophoblasts. During past and chronic infection, COX-2 was highly expressed in trophoblasts (Sarr, personal communication).