TLR4-Endothelin Axis Controls Syncytiotrophoblast Motility and Confers Fetal Protection in Placental Malaria

Abstract

Pregnancy-associated malaria is often associated with adverse pregnancy outcomes. Placental circulatory impairments are an intriguing and unsolved component of malaria pathophysiology. Here, we uncovered a Toll-like receptor 4 (TLR4)-TRIF-endothelin axis that controls trophoblast motility and is linked to fetal protection during Plasmodium infection. In a cohort of 401 pregnancies from northern Brazil, we found that infection during pregnancy reduced expression of endothelin receptor B in syncytiotrophoblasts, while endothelin expression was only affected during acute infection. We further show that quantitative expression of placental endothelin and endothelin receptor B proteins are differentially controlled by maternal and fetal TLR4 alleles. Using murine malaria models, we identified placental autonomous responses to malaria infection mediated by fetally encoded TLR4 that not only controlled placental endothelin gene expression but also correlated with fetal viability protection. In vitro assays showed that control of endothelin expression in fetal syncytiotrophoblasts exposed to Plasmodium-infected erythrocytes was dependent on TLR4 via the TRIF pathway but not MyD88 signaling. Time-lapse microscopy in syncytiotrophoblast primary cultures and cell invasion assays demonstrated that ablation of TLR4 or endothelin receptor blockade abrogates trophoblast collective motility and cell migration responses to infected erythrocytes. These results cohesively substantiate the hypothesis that fetal innate immune sensing, namely, the TRL4-TRIF pathway, exerts a fetal protective role during malaria infection by mediating syncytiotrophoblast vasoregulatory responses that counteract placental insufficiency.

Keywords: Toll-like receptor 4; endothelin; endothelin receptor B; placental malaria; trophoblasts.

FIG 1

Malaria infection during pregnancy reduces birth weight, placental weight, and placental expression of endothelin (EDN) and endothelin receptor B (EDNRB) proteins. Data from 199 female newborns (A) and their placentas (B, C, and D) are plotted according to maternal infection status during pregnancy. NI, uninfected (n = 87); I, infected (n = 112); PV, with P. vivax infection (n = 58); PF, with P. falciparum infection (n = 29); M, with mixed P. vivax/P. falciparum infection (n = 25). (C and D) Percentage of the area of the tissue which stained positively for EDN and EDNRB in immunohistochemistry (IHC)-stained placental sections was quantified using QuPath software. For panels A to D, ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, nonsignificant (adjusted P value of Dunnett’s multiple-comparisons test). (E to H) Correlation of EDN and EDNRB measurements in women who did not have a malaria infection during pregnancy (n = 150) (E), who were infected but had no evidence of PM at the time of delivery (n = 147) (F), who had signs of previous PM (n = 60) (G), and who had active, chronic PM at the time of delivery (n = 10) (H). (I) Correlation was not found in women showing acute PM infection (n = 16). (E to I) Samples were classified according to maternal infection and histopathology criteria as described in Fig. S1 in the supplemental material. P values and Pearson’s correlation coefficient are shown. (J) Ratio of odds of low birth weight in the presence of maternal or placental infection with 95% confidence interval (CI) (acute, P = 0.011; chronic, P = 0.017).

FIG 2

Endothelin (EDN) and endothelin receptor B (EDNRB) protein expression in placentas of female newborns is under TLR4 genetic control. (A) Diagram with relative positioning of 17 SNPs tested in the TLR4 gene. The table shows results of quantitative trait locus analysis testing association of fetal or maternal minor alleles with area of staining of EDN or EDNRB proteins in placental IHC sections of 199 female newborns. MAF, minor allele frequency; P-emp, empirical P value (adjusted with 1 × 10⁶ label permutations). Significant association results are highlighted in bold. (B to D) Genetic effects of maternal alleles at RS11536878 (B) and RS11536879 (C) and RS4986790 (D) fetal alleles (not tested in infected placentas due to low allele frequency). Mann-Whitney P value (D) and Dunn’s posttest P value (B and C) for individual genotype class comparisons.

FIG 3

Placental TLR4 protect fetal viability during placental malaria. (A) Breeding scheme showing Tlr4^−/− females crossed with Tlr4^+/− males to generate heterogenic pregnancies with Tlr₄^+/− and Tlr4^−/− fetuses. Females were infected on the 13th day of gestation, and fetuses and placentas were collected on the 18th day. (B) Maternal TLR4 ablation did not impact on peripheral parasitemia at G₁₈. (C) Premature delivery (prior to G₁₈) in uninfected and infected Tlr₄^+/+ and Tlr4^−/− females (χ² tests; *, P < 0.05; **, P < 0.01). (D) Survival rate of Tlr4^+/+ fetuses in uninfected and infected mothers. Generalized linear mixed-effects model, with infection as fixed effects and litter-wise random effects for fetuses from the same litter; P (infection) < 0.001 (E) Survival rate of Tlr4^+/− fetuses is compared to Tlr4^−/− counterparts. Generalized linear mixed-effects model, with infection and fetal Tlr4 genotype as fixed effects and litter-wise random effects for fetuses from the same litter; P (Tlr₄^+/−) = 0.037; P (infection) = 0.049. (F) Correlation of litter viability rate with proportion of Tlr₄^+/− fetuses in litter (each data point represents one heterogenic litter; n = 10).

FIG 4

Fetal TLR4 modulates placental endothelin 1 (Edn1) gene expression during malaria infection. Placental expression of Edn1 and Ednrb using one Tlr4-discordant sibling pair from each heterogenic litter. (A to D) Grouping by fetal genotype to evaluate the effect of infection represented by fold change relative to the average of noninfected placentas. (E to H) Grouping by infection status to evaluate the effect of placental Tlr4 genotype represented by fold change relative to the average of Tlr4^−/− (pairwise analysis). (I and J) Images of EDN (I) and EDNRB (J) immunohistochemistry staining showing the pattern of protein expression throughout the placental labyrinth. Scale bar, 100 μm. (K) Parasite load in Tlr₄^+/− and Tlr4^−/− placentas (pairwise analysis) from infected females. (A to D) Wilcoxon rank-sum (Mann-Whitney) tests. n = 6 per group; **, P < 0.01. (E to H and K) Wilcoxon matched-pairs signed-rank tests, n (noninfected) = 8 sibling pairs, n (infected) = 10 sibling pairs; **, P < 0.01.

FIG 5

Edn1 expression is downregulated in vitro upon trophoblast-IE interactions in a TLR4- and TRIF-dependent manner. (A to D) Edn1 expression in primary murine trophoblasts from wild-type (A), Tlr4^−/− (B), Myd88^−/− (C), and Trif^−/− (D) mice exposed to infected erythrocytes (IEs) for 4 h. Data represented as fold change relative to exposure to noninfected erythrocytes (NIEs). Ednrb expression in wild-type (E) or Tlr4^−/− (F) primary murine trophoblasts exposed to IEs or NIEs for 4 h. Each data point represents the average value of experimental triplicates. Mann-Whitney tests; *, P < 0.05; **, P < 0.01. Plots representative of multiple experiments.

FIG 6

Exposure to infected erythrocytes reduces trophoblast motile activity in a TLR4-dependent manner. Time lapse imaging (Movies S1 and S2 in the supplemental material) of primary trophoblasts was carried out in 9 adjacent microscope fields over a 4-h period (10-min intervals), and overall trophoblast conformation activity was measured between consecutive frames (ΔRID). (A) Representative plot of the ΔRID data obtained from one experiment, showing the ARID values of three replicates exposed to infected erythrocytes (IEs) compared to noninfected erythrocytes (NIEs), indicating reduced collective motility. (B) Area under the curve (AUC), representing the total amount of movement over the 4-h period, was calculated from the ΔRID data of each culture well across 5 independent experiments, and each point was then standardized to the average of the uninfected controls. Ratio-paired t test was applied to the mean AUCs of each experiment, **, P < 0.01. (C) Trophoblast migration assays were carried out with primary trophoblasts seeded on a ECM gel cushion in a Transwell. (D and E) Number of cells that crossed the layer was quantified and is reduced in wild-type trophoblasts which are exposed to IEs (D), but not in exposed Tlr4^−/− trophoblasts (E). (F) Addition of the endothelin receptor antagonist, bosentan, also reduces migration in primary wild-type trophoblasts. One-sample t tests; *, P < 0.05; **, P < 0.01.