Involvement of Th1Th17 Cell Subpopulations in the Immune Responses of Mothers Who Gave Birth to Children with Congenital Zika Syndrome (CZS)

Abstract

High levels of T helper 17 cell (Th17)-related cytokines have been shown in acute Zika virus (ZIKV) infection. We hypothesized that the high levels of Th17-related cytokines, associated with a regulatory environment during pregnancy, create a favorable milieu for the differentiation of CD4+Th17 cells. We present data from a cross-sectional study on mothers who confirmed ZIKV infection by qRT-PCR and their children. We also recruited non-pregnant women infected with ZIKV in the same period. ZIKV infection occurred between 2015 and 2017. We collected samples for this study between 2018 and 2019, years after the initial infection. We highlight that, after in vitro stimulation with ZIKV CD4 megapool (ZIKV MP), we found a lower frequency of IL-17-producing CD4+ T cells (Th17), especially in the mothers, confirmed by the decrease in IL-17 production in the supernatant. However, a higher frequency of CD4+ IL-17+ IFN-γ+ T cells (Th1Th17) responding to the ZIKV MP was observed in the cells of the mothers and children but not in those of the non-pregnant women. Our data indicate that the priming of CD4 T cells of the Th1Th17 phenotype occurred preferentially in the mothers who gave birth to children with CZS and in the children.

Keywords: T cells; Th17 cells; Zika; congenital Zika syndrome (CZS); pregnancy.

Figure 1

Subpopulations of CD4+ T cell phenotypes among individuals with a history of ZIKV infection. CD4 ZIKV-restricted responses among women (from non-pregnant women infected with ZIKV, violet circles, n = 6), mothers (from pregnant mothers infected with ZIKV, blue squares, n = 13) and children (from children born to mothers infected with ZIKV during pregnancy, red triangles, n = 13) with histories of ZIKV infection, after 6 h of in vitro stimulation with ZIKV MP. (A) Gating strategy for the flow cytometry plots identifying ZIKV-specific CD4+ T cells. Black arrows indicate the step-by-step analysis. (B) Percentages of CD4+ T cells that express CD45RA (RA+) and that do not express CD45RA (RA–). (C) Percentage of naive CD4 T cell subsets (Tn: CD45RA+CCR7+); (D) central memory (Tcm: CD45RA–CCR7+); (E) effector memory cells (Tem: CD45RA–CCR7–) and (F) effector memory RA T cells (Temra: CD45RA+CCR7–). (B) Differences between CD45RA+ and CD45RA– CD4+ T cells were analyzed within each group using a Wilcoxon matched-pairs signed rank test. In addition, differences in the frequencies of each RA+ or RA–among the groups were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (C–F) Differences in subsets of CD4+ T cells among the groups were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data are expressed as the mean with standard deviation for each group. Each data point represents a single individual determination. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Subpopulations of Th1 and Th2 CD4+ T cell phenotypes among individuals with a history of ZIKV infection. Th1 and Th2 CD4+ subsets were measured in women (from non-pregnant women infected with ZIKV, violet circles, n = 6), mothers (from pregnant mothers infected with ZIKV, blue squares, n = 16) and children (from children born to mothers infected with ZIKV during pregnancy, red triangles, n = 6) with histories of ZIKV infection without stimulation. (A) Representative gating of CCR4 and CXCR3 among CD4+ cells that do not express either CCR6 or CD45RA from donor PBMCs is shown. Th1 cells were identified from CCR4− CXCR3+ and Th2 from CCR4+ CXCR3−. In other staining, we gated CD127+ CD4+ T cells with the specific Th1 transcription factor T-bet, and Th2 cells for GATA-3. Black arrows indicate the step-by-step analysis. (B) Percentage of Th1 CD4+ cells from surface markers and from transcription factor T-bet among the groups. (C) Percentage of Th2 CD4+ cells from surface markers and from transcription factor GATA-3 among the groups. (B,C) Differences in the frequencies of Th1 and Th2 CD4+ cells from surface markers were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Furthermore (B,C), differences in the frequencies of Th1 or Th2 CD4+ cells from intracellular staining of T-bet and GATA-3 were analyzed using the Mann–Whitney test. Data are expressed as the mean with standard deviation for each group. Each data point represents a single individual determination. Asterisks indicate significant differences (* p < 0.05).

Figure 3

Subpopulations of Th17, Th1Th17, R6+DN and R6+DP CD4+ T cell phenotypes among individuals with a history of ZIKV infection. Th17 CD4+ subsets were measured in women (from non-pregnant women infected with ZIKV, violet circles, n = 6), mothers (from pregnant mothers infected with ZIKV, blue squares, n = 16) and children (from children born to mothers infected with ZIKV during pregnancy, red triangles, n = 6) with histories of ZIKV infection without stimulation. (A) Representative gating of CCR4 and CXCR3 among CD4+ cells that express CCR6 but do not express CD45RA from donor PBMCs is shown. Th17 cells were identified from CCR4+ CXCR3−, Th1Th17 from CCR4− CXCR3+, R6+DN from CCR4− CXCR3− and R6+DP from CCR4+ CXCR3+. Black arrows indicate the step-by-step analysis. Percentages of (B) Th17 (C) Th1Th17, (D) R6+DP and (E) R6+DN CD4+ cells among the groups. (F) Relative contribution of each Th17 subset within each group. (B–F) Differences in the frequencies of Th17 CD4+ subsets were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. In (F), differences in the frequencies of Th17 subsets between the groups were analyzed using the Friedman test followed by Dunn’s multiple comparisons test. Data are expressed as the mean with standard deviation for each group. Each data point represents a single individual determination. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 4

Frequency of memory CD4+ CCR6+ subsets after stimulation with ZIKV MP in individuals with a history of ZIKV infection. Memory CCR6+ and CCR6− CD4+ subsets were measured in women (from non-pregnant women infected with ZIKV, violet circles, n = 6), mothers (from pregnant mothers infected with ZIKV, blue squares, n = 8) and children (from children born to mothers infected with ZIKV during pregnancy, red triangles, n = 6) with histories of ZIKV infection after ZIKV MP stimulation. (A) Representative gating of CCR6 among CD4+ cells according to CD45RA− and/or CCR7+ expression from donor PBMCs is shown. Black arrows indicate the step-by-step analysis. In addition, R6− or R6+EM/TM cells were identified through the absence of CCR7 expression and R6− or R6+CM cells expressing CCR7. (B) Percentages of R6− EM/TM and R6−CM. (C) Percentages of R6+ EM/TM and R6+CM. Differences between cells stimulated with ZIKV MP and unstimulated cells were analyzed using the Wilcoxon matched-pairs signed rank test. Each data point represents a single individual determination without (unstimulated) and after stimulation with ZIKV MP. Grey bars represent the mean for each group. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01).

Figure 5

Levels of IL-17A, IL-6 and TGF-β after stimulation with ZIKV MP in individuals with a history of ZIKV infection. Cytokine production in culture supernatants without stimulation or after 20 h of stimulation with ZIKV MP were quantified by means of ELISA. The data represent women (from non-pregnant women infected with ZIKV, violet circles, n = 5), mothers (from pregnant mothers infected with ZIKV, blue squares, n = 19) and children (from children born to mothers infected with ZIKV during pregnancy, red triangles, n = 17) with histories of ZIKV infection. (A) IL-17 levels among the groups: between mothers who had asymptomatic babies (n = 9) and those who had babies with CZS (n = 10), and between asymptomatic children (n = 11) and those born with CZS (n = 6). Similarly, for (B) IL-6 and (C) TGF-β. Differences between cells stimulated with ZIKV MP and unstimulated cells were analyzed using the Wilcoxon matched-pairs signed rank test. Each data point represents a single individual determination without (unstimulated) and after stimulation with ZIKV MP. Grey bars represent the mean for each group. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

Frequencies of the responding IL-17- and IL-17+ IFN-γ+-producing CD4+ T cell subsets in individuals with histories of ZIKV. Cytokine-producing CD4+ T cells were measured in women (from non-pregnant women infected with ZIKV, violet circles, n = 6), mothers (from pregnant mothers infected with ZIKV, blue squares, n = 21) and children (from children born to mothers infected with ZIKV during pregnancy, red triangles, n = 12) with histories of ZIKV infection, after stimulation with ZIKV MP. (A) Representative gating of IL-17+ IFN-γ− and IL-17+ IFN-γ+-producing CD4+ T cell subsets from donor PBMCs is shown. Percentages of (B) IL-17+ IFN-γ− and (C) IL-17+ IFN-γ+-producing CD4+ T cell subsets in each group. Cytokine-producing CD4+ T cells were also evaluated between the mothers who had asymptomatic babies (n = 11) and those who had babies with CZS (n = 10) and between the asymptomatic children (n = 6) and those born with CZS (n = 6). Differences between cells stimulated with ZIKV MP and unstimulated cells were analyzed using the Wilcoxon matched-pairs signed rank test. Each data point represents a single individual determination without (unstimulated) and after stimulation with ZIKV MP. Grey bars represent the mean for each group. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).