Peripheral blood cell signatures of Plasmodium falciparum infection during pregnancy

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 3c1000 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

Peripheral blood cell signatures of Plasmodium falciparum infection during pregnancy

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

Characteristics of sub-groups compared with the whole cohort in Benin at inclusion and at delivery.

<p>Values are means (standard deviation) except for parasitaemia which are medians (interquartile ranges).</p>*<p>Student t test or χ² for proportions.</p>†<p>3 subjects were positive by RDT but negative by microscopy.</p>‡<p>4 subjects were positive by RDT but negative by microscopy.</p

<i>P. falciparum</i> infection-related changes in peripheral blood mononuclear cell frequencies of pregnant Beninese at inclusion and at delivery.

<p>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.</p

Cytometry-based gating for definition of phenotypes.

<p>(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⁻).</p

Multiple logistic regression analysis for independent associations of selected parameters with the presence of <i>P. falciparum</i> infection in the Benin sub-group at delivery.

a<p>median value of uninfected group used for dichotomisation.</p

Multiple logistic regression analysis for independent associations of selected parameters with the presence of <i>P. falciparum</i> infection in the Benin sub-group at inclusion.

a<p>median value of uninfected group used for dichotomisation.</p><p>χ² Hosmer-Lemeshow test (15ddl) = 13.9, p = 0.53.</p

Multiple logistic regression analysis for independent associations of selected parameters with the presence of anaemia in the Benin sub-group at inclusion.

a<p>median value of non-anaemic group used for dichotomisation.</p><p>χ² Hosmer-Lemeshow test (17ddl) = 7.91; p = 0.97.</p