Peripheral blood cell signatures of Plasmodium falciparum infection during pregnancy

Abstract

Sequestration of Plasmodium falciparum-infected erythrocytes in placental intervillous spaces causes inflammation and pathology. Knowledge of the profiles of immune cells associated with the physiopathology of pregnancy-associated malaria (PAM) is scarce. We conducted a longitudinal, prospective study, both in Benin and Tanzania, including ∼1000 pregnant women in each site with systematic follow-up at scheduled antenatal visits until delivery. We used ex vivo flow cytometry to identify peripheral blood mononuclear cell (PBMC) profiles that are associated with PAM and anaemia, determining the phenotypic composition and activation status of PBMC in selected sub-groups with and without PAM both at inclusion and at delivery in a total of 302 women. Both at inclusion and at delivery PAM was associated with significantly increased frequencies both of B cells overall and of activated B cells. Infection-related profiles were otherwise quite distinct at the two different time-points. At inclusion, PAM was associated with anaemia, with an increased frequency of immature monocytes and with a decreased frequency of regulatory T cells (Treg). At delivery, infected women presented with significantly fewer plasmacytoid dendritic cells (DC), more myeloid DC expressing low levels of HLA-DR, and more effector T cells (Teff) compared to uninfected women. Independent associations with an increased risk of anaemia were found for altered antigen-presenting cell frequencies at inclusion, but for an increased frequency of Teff at delivery. Our findings emphasize the prominent role played by B cells during PAM whenever it arises during pregnancy, whilst also revealing signature changes in other circulating cell types that, we conclude, primarily reflect the relative duration of the infections. Thus, the acute, recently-acquired infections present at delivery were marked by changes in DC and Teff frequencies, contrasting with infections at inclusion, considered chronic in nature, that were characterized by an abundance of immature monocytes and a paucity of Treg in PBMC.

Figure 1. Cytometry-based gating for definition of phenotypes.

(A) Monocytes (CD14⁺), B cells (CD19⁺) were gated from PBMC. pDC were directly gated from the CD14⁻CD19⁻ population whilst mDC were gated from the CD14⁻CD19⁻HLA-DR⁺ population. CD86 and HLA-DR expression were determined by MFI (Mean Fluorescence Intensity). (B) NK, NK T and CD3⁺ cells were gated from PBMC. CD4⁺ and CD8⁺ T cells were gated from CD3⁺. The gating strategies for Treg (CD4⁺CD25⁺CD127⁻) and Teff (CD4⁺CD25⁺CD127⁺) are presented in (C). Cell frequencies were determined as a percentage of PBMC, and relative FoxP3 expression level determined as a function of FoxP3 expresssion by naïve CD4⁺ T cells (CD4⁺CD25⁻).

Figure 2. Basic flow cytometry-based phenotypic characterization of different cell populations investigated.

The left panel lists the cell types identified with different surface markers and the right panel lists the separate markers used to characterize antigen-presenting cells activation status (HLA-DR & CD86) and to distinguish CD4⁺ regulatory T cells (Treg, expressing the transcription factor FoxP3) from effector T cells (Teff).

Figure 3. P. falciparum infection-related changes in peripheral blood mononuclear cell frequencies of pregnant Beninese at inclusion and at delivery.

Scatter plots include bars depicting medians with interquartile ranges of antigen-presenting cells (A, B, C and D) and T cell subset frequencies (E, F, G and H) from 69 uninfected compared to 62 infected women at inclusion, and from 47 uninfected, 27 exposed and 37 infected women at delivery. The statistical significance of differences between profiles segregated according to the presence or absence of infection were determined using the non-parametric Mann Whitney U test for data at inclusion combined with the non-parametric Kruskall Wallis test for data at delivery. *p<0.05, **p<0.01.

Figure 4. P. falciparum infection-related changes in activation status of antigen-presenting cells at inclusion and delivery in pregnant Beninese.

Scatter plots include bars depicting medians with interquartile ranges of HLA-DR expression (A–D) and CD86 expression (E–H) on APC of 69 uninfected compared to 62 infected women at inclusion and of 47 uninfected, 27 exposed and 37 infected women at delivery. The statistical significance of differences in levels of expression were determined using the non-parametric Mann Whitney U test for data at inclusion combined with the Kruskall Wallis test for data at delivery. *p<0.05, **p<0.01, ***p<0.001.