Plasmodium falciparum infection dysregulates placental autophagy

Abstract

Plasmodium (P.) falciparum malaria during pregnancy has been frequently associated with severe consequences such as maternal anemia, abortion, premature birth, and reduced birth weight. Placental damage promotes disruption of the local homeostasis; though, the mechanisms underlying these events are still to be elucidated. Autophagy is a fundamental homeostatic mechanism in the natural course of pregnancy by which cells self-recycle in order to survive in stressful environments. Placentas from non-infected and P. falciparum-infected women during pregnancy were selected from a previous prospective cohort study conducted in the Brazilian Amazon (Acre, Brazil). Newborns from infected women experienced reduced birth weight (P = 0.0098) and placental immunopathology markers such as monocyte infiltrate (P < 0.0001) and IL-10 production (P = 0.0122). The placentas were evaluated for autophagy-related molecules. As a result, we observed reduced mRNA levels of ULK1 (P = 0.0255), BECN1 (P = 0.0019), and MAP1LC3B (P = 0.0086) genes in placentas from P. falciparum-infected, which was more striking in those diagnosed with placental malaria. Despite the protein levels of these genes followed the same pattern, the observed reduction was not statistically significant in placentas from P. falciparum-infected women. Nevertheless, our data suggest that chronic placental immunopathology due to P. falciparum infection leads to autophagy dysregulation, which might impair local homeostasis during malaria in pregnancy that may result in poor pregnancy outcomes.

Fig 1. Effect of P. falciparum infection during pregnancy in placental autophagy-related genes expression.

(A-C) Placental mRNA expression of the selected autophagy-related genes ULK1 (A), BECN1 (B), and MAP1LC3B (C) in non-infected (NI) and P. falciparum-infected (Pf-INF) women. Results represent qPCR estimates relative to NI and normalized by GAPDH, endogenous control. Data sets on placental mRNA levels from P. falciparum-infected women were afterwards stratified according to placental malaria (PM) status as placental malaria negative (Pf-INF PM-) or positive (Pf-INF PM+) and plotted for ULK1 (D), BECN1 (E), and MAP1LC3B (F). (G) Heatmap matrixes of NI and Pf-INF placentas pairwise Spearman correlation coefficients (r_s) between mRNA levels and histologic and immunologic parameters. Data are represented as whiskers boxplots where the bottom and the top of the box are the first and third quartiles, the line inside the box is the median and the whiskers represent the minimum and the maximum values (A-F), and heatmaps containing r_s in a colour range from dark blue (r_s = -1) to dark red (r_s = 1) with statistically significant correlations enhanced as bold values (G). The differences between groups were determined by the Mann-Whitney U test (A-C), Kruskal-Wallis test with Dunn’s post-test for multiple comparisons (D-F), and Spearman’s rank-order non-parametric test for correlations (G). P values < 0.05 were considered as representing statistically significant differences. BW–birth weight, PW–placental weight, HZ–hemozoin, SNA–syncytial nuclear aggregates, NECR–necrosis, VASC–vascularity, LEU–leukocytes, MO–monocytes.

Fig 2. Levels of the autophagy-related proteins are diminished in P. falciparum-infected placentas.

Placental autophagy-related protein levels measured in non-infected (NI) and P. falciparum-infected (Pf-INF) women. (A) Representative image of cropped western blotting of ULK1 (120 kDa), BECLIN1 (52 kDa), LC3I, and LC3II (16 and 14 kDa, respectively), and corresponding β-ACTIN (42 kDa) (full-length blots at S1 Fig). Western blot was performed at two to three independent experiments. (B-D) Semi-quantification by densitometry analysis of the ULK1 (B), BECLIN1 (C), and LC3II (D) protein levels. Semi-quantification of the target proteins was performed in different blots without image manipulation and normalized to the corresponding β-ACTIN (endogenous control), which blotting was conducted in the same membrane. Data sets on placental autophagy protein levels from P. falciparum-infected women were afterwards stratified according to placental malaria (PM) status as placental malaria negative (Pf-INF PM-) or positive (Pf-INF PM+) and plotted for ULK1 (E), BECLIN1 (F), and LC3II (G). Data are represented as whiskers boxplots where the bottom and the top of the box are the first and third quartiles, the line inside the box is the median, and the whiskers represent the minimum and the maximum values (B-G). Statistical analysis was performed using the Mann-Whitney U test (B-D) or Kruskal-Wallis test with Dunn’s post-test for multiple comparisons (E-G). P values < 0.05 were considered as representing statistically significant differences.