Study population
Table 1 shows the characteristics of the participants involved in the study. Median age was highest for NNP (median [IQR] of 36.5 [29.6 ; 44] years) and lowest for EP (25.2 [21 ; 29] years) and there were males only in the NNP group. All samples in the NP group were collected at delivery, and the gestational age from NP women was unknown.
Table 1. Description of the study participants.
|
ENP
|
EP
|
NNP
|
NP
|
Characteristics
|
N
|
38
|
154
|
23
|
16
|
Age, years median (IQR)
|
26.5 (20 ; 29.5)
|
25.2 (21 ; 29)
|
36.5
(29.6 ; 44)
|
29
(26.5 ; 32.1)
|
Sex, n (%)
|
Females
|
38 (100)
|
154 (100)
|
9 (39.1)
|
16 (100)
|
Plasmodium infection in peripheral blood, n (%)
|
3 (7.69)
|
27 (13.8)
|
na
|
na
|
Gestational Age,
weeks median (IQR)
|
na
|
30.9 (22.8-40)
|
na
|
nd
|
Sampling timepoint, n (%)
|
Recruitment (peripheral blood)
|
na
|
54 (35.1)
|
na
|
0 (0)
|
Delivery (peripheral and placental blood)
|
na
|
100 (64.9)
|
na
|
16 (100)
|
Abbreviations: ENP, exposed non-pregnant; EP, exposed pregnant; NNP, naïve non-pregnant; NP, naïve pregnant; na, not applicable; nd, not determined; IQR, Interquartile range.
Correlations of peripheral plasma concentrations of eotaxin-1, eotaxin-2 and eotaxin-3
There was a weak positive but significant correlation between eotaxin-1 and eotaxin-2 levels (Spearman’s rho (R) = 0.25, p < 0.05) and eotaxin-2 and eotaxin-3 (R = 0.35, p < 0.05), whereas there was no correlation between eotaxin-1 and eotaxin-3 (Figure 1).
Differences in eotaxin-2 and -3 peripheral plasma concentrations by malaria exposure and pregnancy
There were significant differences in the eotaxin-2 concentrations between groups (p ≤ 0.001). When we compared groups two-by-two, we did not find statistical differences in the plasma concentrations associated to pregnancy (NNP vs NP or ENP vs EP, Figure 2A). However, significantly lower eotaxin-2 plasma concentrations were observed in malaria-exposed groups, both in the non-pregnant (NNP vs ENP) and the pregnant (NP vs EP) individuals (Figure 2A). Eotaxin-3 plasma levels followed the same pattern as eotaxin-2 i.e., there were significant differences among the four study groups (p ≤ 0.001) and malaria-exposure, but not pregnancy, was associated with lower levels of eotaxin-3 plasma concentrations (Figure 2B).
Correlations of eotaxin-2 and eotaxin-3 peripheral plasma concentrations with markers of malaria exposure
To further investigate the association between eotaxin-2 and -3 and Plasmodium exposure (P. falciparum or P. vivax), the correlations between the concentrations of these two eotaxins and IgG levels against 19 malaria antigens in peripheral plasma were assessed in the malaria-exposed group (Table 2). Most of the IgGs analyzed are known markers of malaria exposure (like IgG to PfAMA-1) or malaria in pregnancy (MiP) (like IgG to PfDBL5e or PfDBL3x). The greatest correlations observed were: PfDBL5e-IgG with eotaxin-2 and -3 (REo2 = - 0.28 p=0.038, REo3 = - 0.37 p=0.011) and PfDBL3x-IgG with eotaxin-3 (REo3 = - 0.36 p=0.011) (Table 2), each of them shown as scatterplot in Figure 3A.
Table 2. Correlations of eotaxin-2 and eotaxin-3 peripheral plasma concentrations with malaria IgG levels.
|
Eo2
|
Eo3
|
PfMSP-119
|
0.07
|
0.04
|
PfEBA
|
0.02
|
0.05
|
PfDBL6ε
|
-0.11
|
-0.04
|
PfDBL5ε
|
-0.28
|
-0.37
|
PfDBL3x
|
-0.17
|
-0.36
|
PfAMA-1
|
0.04
|
-0.19
|
PvLP2
|
0.10
|
-0.01
|
PvLP1
|
0.11
|
-0.11
|
Vir14
|
-0.07
|
-0.07
|
Vir2/15
|
-0.02
|
-0.04
|
Vir25
|
0.04
|
-0.10
|
PvMSP-5
|
0.21
|
0.00
|
Pv200L
|
-0.09
|
-0.02
|
PvMSP-119
|
-0.02
|
-0.10
|
PvDBP
|
0.06
|
0.02
|
PvCSP-R
|
0.15
|
0.19
|
PvCSP-N
|
-0.16
|
0.00
|
PvCSP-C
|
0.07
|
0.11
|
PvCSP
|
0.04
|
0.02
|
Color scale ranges between the red (R=1) and the purple (R=-1), and the specific Spearman’s coefficient is displayed in the cells. Only the malaria exposed study groups are included in the analysis (n=78). In bold if p<0.05. Eo: Eotaxin
We next investigated the correlation between eotaxin concentrations in peripheral plasma and the frequency of some B cell subsets, also known to be altered in malaria-exposed individuals and analyzed in the same women as part of a previous study (6). There was a significant negative correlation between the frequency of atypical MBC and the concentrations of both chemokines, especially eotaxin-2 (Table 3). A weaker but significant negative correlation was also observed between eotaxin-2 concentration and active classical MBCs. In contrast, MZ-MBCs showed a positive and significant correlation with plasma concentration of eotaxin-2. A scatter plot showing each of the strongest correlations is shown in Figure 3B.
Table 3. Correlations of eotaxin-2 and eotaxin-3 peripheral plasma concentrations with B cell subsets frequencies.
|
Eo2
|
Eo3
|
MZ-MBC
|
0.33
|
0.1
|
Naïve
|
0.03
|
0.08
|
rcMBC
|
0.09
|
-0.01
|
aaMBC
|
-0.37
|
-0.28
|
acMBC
|
-0.21
|
-0.07
|
Color scale ranges between the red (R=1) and the purple (R=-1) and the specific Spearman’s coefficient is displayed in the cells. The four study groups are included: NNP (n=23), NP (n=13), ENP (n=38) and EP (n=82). In bold if p<0.05. MBC memory B cells: MZ-MBC: marginal zone-like MBC, rcMBC: resting classical MBC, aaMBC: active atypical MBC, acMBC: active classical MBC, Eo: Eotaxin.
Associations between eotaxin-2 and eotaxin-3 peripheral plasma concentrations and malaria infectionRegression models were estimated to evaluate the possible associations between eotaxin-2 and/or eotaxin-3 plasma concentrations and Plasmodium infection (P. falciparum or P. vivax), age, hemoglobin levels and gestational age (in the pregnant study groups). Neither age nor hemoglobin levels had an association or trend with eotaxin-2 or -3 concentrations (data not shown), but gestational age was significantly associated with eotaxin-3 concentration being higher at delivery (Table 4, Coeff = 3.161; 95%CI: 0.322; 6.000, p= 0.031). Interestingly, in the exposed pregnant women, eotaxin-3 showed a trend of negative association with Plasmodium infection at recruitment in the crude analysis and after adjusting by gestational age (Table 4, adjusted effect estimate, Coeff = -0.279; 95%CI: -0.605 - 0.047, p = 0.091), but no association was observed at delivery. Because the prevalence of infection was relatively low at enrolment and delivery (14.19%), crude and adjusted regression models were also estimated with both pregnant groups together, but no effect was seen (Table 4).
Table 4. Associations between plasma eotaxin-2 and eotaxin-3 concentrations and gestational age or Plasmodium infection.
|
Eotaxin-2
|
Eotaxin-3
|
|
Coefficient
|
95% CI
|
p-value
|
Coefficient
|
95% CI
|
p-value
|
Gestational Age
|
Recruitment
|
0.159
|
-0.343 ; 0.662
|
0.526
|
0.064
|
-0.229 ; 0.357
|
0.662
|
Delivery
|
0.430
|
-3.321 ; 4.182
|
0.815
|
3.161
|
0.322 ; 6.000
|
0.031
|
EP
|
-0.012
|
-0.563 ; 0.539
|
0.960
|
-0.094
|
-0.407 ; 0.218
|
0.541
|
Plasmodium Infection
|
Recruitment
|
-0.218
|
-0.822 ; 0.386
|
0.471
|
-0.283
|
-0.601 ; 0.035
|
0.079
|
Delivery
|
-0.433
|
-1.329; 0.465
|
0.331
|
-0.256
|
-0.935 ; 0.424
|
0.44
|
EP
|
-0.467
|
-1.213 ; 0.279
|
0.213
|
-0.147
|
-0.570 ; 0.284
|
0.49
|
Recruitment*
|
-0.202
|
-0.813 ; 0.409
|
0.509
|
-0.279
|
-0.605 ; 0.047
|
0.091
|
Delivery*
|
-0.470
|
-1.402 ; 0.461
|
0.308
|
-0.454
|
-1.057 ; 0.149
|
0.130
|
EP*
|
-0.508
|
-1.299 ; 0.283
|
0.202
|
-0.121
|
-0.576 ; 0.333
|
0.588
|
Crude or adjusted by gestational age (*) linear regression models were estimated with eotaxin-2 and eotaxin-3 as dependent variables and gestational age or Plasmodium spp. infection as independent variables, for the malaria exposed pregnant women group EP (n = 81), including recruitment (N=54) and delivery (N=27) timepoints. Plasmodium infection included P. vivax or P. falciparum infections. Bold = p-value < 0.1.
Eotaxin-2 concentration in peripheral and placental plasma in malaria exposed and non-exposed pregnant women.
We had previously hypothesized that placental eotaxin-1 may be decreased in placenta compared to periphery in a tropical cohort of pregnant women (22), to avoid receptor competition with eotaxin-2, shown to enhance decidualization in vitro (6). Thus, we measured eotaxin-2 levels in placental plasma samples separately in malaria-exposed and malaria-naive women. We found an important and significant decrease in eotaxin-2 placental plasma concentrations compared with peripheral plasma concentrations in malaria-naïve women (Figure 4A), while an increase was observed in malaria-exposed women (Figure 4B). Also, there was a trend for increased eotaxin-2 levels in placental plasma samples of malaria-exposed women compared with malaria-naïve (Figure 5).