Investigation of Plasma-Derived Lipidome Profiles in Experimental Cerebral Malaria in a Mouse Model Study

Abstract

Cerebral malaria (CM), a fatal complication of Plasmodium infection that affects children, especially under the age of five, in sub-Saharan Africa and adults in South-East Asia, results from incompletely understood pathogenetic mechanisms. Increased release of circulating miRNA, proteins, lipids and extracellular vesicles has been found in CM patients and experimental mouse models. We compared lipid profiles derived from the plasma of CBA mice infected with Plasmodium berghei ANKA (PbA), which causes CM, to those from Plasmodium yoelii (Py), which does not. We previously showed that platelet-free plasma (18k fractions enriched from plasma) contains a high number of extracellular vesicles (EVs). Here, we found that this fraction produced at the time of CM differed dramatically from those of non-CM mice, despite identical levels of parasitaemia. Using high-resolution liquid chromatography-mass spectrometry (LCMS), we identified over 300 lipid species within 12 lipid classes. We identified 45 and 75 lipid species, mostly including glycerolipids and phospholipids, with significantly altered concentrations in PbA-infected mice compared to Py-infected and uninfected mice, respectively. Total lysophosphatidylethanolamine (LPE) levels were significantly lower in PbA infection compared to Py infection and controls. These results suggest that experimental CM could be characterised by specific changes in the lipid composition of the 18k fraction containing circulating EVs and can be considered an appropriate model to study the role of lipids in the pathophysiology of CM.

Keywords: Plasmodium spp.; cerebral malaria; lipidome; mouse model.

Figure 1

Lipid classes in plasma microvesicles (MV) from uninfected controls, PbA-infected and Py-infected mice. (A) All lipid classes. (B) Levels without triglycerides (TG) and cholesteryl esters (CE).

Figure 2

Altered lipid class levels in MV (18k PFP) from the three groups of mice. The mean value of the concentrations (pmol/uL) of lipid ions belonging to a lipid class was estimated. The lipid ion concentration was calculated by normalising to the respective internal standards (A) Boxplots comp the distribution statistics (the minimum, maximum, median and the first and third quartiles) of the 12 lipid classes stratified to the groups of control (green), PbA (red) and Py (blue). Each dot corresponds to a sample, with its value representing the mean concentration of lipid ions of the respective lipid class. Data were log-transformed. (B) Significance of differences was assessed using Student’s t-test followed by FDR correction; adjusted p-values of 0.05 and 0.1 are marked with dashed and solid lines, respectively. Abbreviations as in Figure 1.

Figure 3

Differentially expressed lipids. (A) Principal component analysis (PCA) of identified lipids (pmol/µL). (B) Volcano plots showing differentially expressed lipids in the various pairwise comparisons. (C) Bar charts representing the proportion of differentially expressed ions across each lipid graph. Blue, grey and red bars show the percentage of elevated, not significant and decreased level of lipid molecules in each type. Numbers on top show the actual number of differentially expressed ions in each type. (D) Venn diagram of the distribution of differentially expressed lipids in the various comparisons. Significance is based on two-fold increase or decrease in lipid amounts plus an adjusted p-value < 0.01 based on moderated t-test and false discovery rate (FDR) correction.

Figure 4

Docosahexaenoic acid (DHA 22:6) containing TG lipids are higher during cerebral malaria (CM). Comparison of levels of characterised TG species from plasma MVs between PbA (red) vs. control (green) and Py (blue). Yellow boxes highlight DHA 22:6 containing TG lipids that are exclusively elevated in PbA vs. control and Py. To display the statistical significance of differences, symbols (-) represents no statistical difference, (#) for PbA vs. control and (*) for PbA vs. Py based on adjusted p-value < 0.01. There are no differences in TGs between Py and control.

Figure 5

Correlation plot of the 121 retained lipids across all the samples. (A) Fold changes (FC) of lipid amounts in the three categories of comparisons. Green denotes an increase and red a decrease. (B) Hierarchical clustering of the lipid–lipid correlation matrix, where the pairwise Pearson correlation was conducted across all samples in the three categories. Rows and columns correspond to the 121 retained lipid species. Dark blue triangles indicate clusters (1–8) of strongly positively correlated lipids. Cluster numbers and corresponding lipid names are shown on the right.