Monocytes from Uninfected Neonates Born to Trypanosoma cruzi-Infected Mothers Display Upregulated Capacity to Produce TNF-α and to Control Infection in Association with Maternally Transferred Antibodies

Abstract

Activated monocytes/macrophages that produce inflammatory cytokines and nitric oxide are crucial for controlling Trypanosoma cruzi infection. We previously showed that uninfected newborns from T. cruzi infected mothers (M+B- newborns) were sensitized to produce higher levels of inflammatory cytokines than newborns from uninfected mothers (M-B- newborns), suggesting that their monocytes were more activated. Thus, we wondered whether these cells might help limit congenital infection. We investigated this possibility by studying the activation status of M+B- cord blood monocytes and their ability to control T. cruzi in vitro infection. We showed that M+B- monocytes have an upregulated capacity to produce the inflammatory cytokine TNF-α and a better ability to control T. cruzi infection than M-B- monocytes. Our study also showed that T. cruzi-specific Abs transferred from the mother play a dual role by favoring trypomastigote entry into M+B- monocytes and inhibiting intracellular amastigote multiplication. These results support the possibility that some M+B- fetuses may eliminate the parasite transmitted in utero from their mothers, thus being uninfected at birth.

Keywords: Trypanosoma cruzi-specific antibodies; congenital chagas disease; monocytes.

Figure 1

TNF-a expression by monocytes from M+B- and M-B-newborns. (A) Purified cord blood monocytes were cultured for 4 h with a T. cruzi trypomastigote lysate (10⁶ parasites/mL, parasite to cell ratio of 1) or LPS (0.01 µg/mL), in the presence of brefeldin (10 µg/mL). The proportion of TNF-a-positive CD14+ cells and intracellular TNF-a levels (shown as mean fluorescence intensities—MFI) were evaluated by flow cytometry. Results are expressed as mean ± SEM of 9 M-B- and 11 M+B- samples. (B) In other similar experiments in which brefeldin was not added, TNF-a was measured in supernatants by ELISA. Results are expressed as mean ± SEM (n = 11 M-B- samples and 14 M+B- samples). *: p < 0.05, **: p < 0.005, ****: p< 0.0001 vs. M-B- group (Mann–Whitney test).

Figure 2

Initial parasite burden of monocytes from M+B- and M-B-newborns, infected with T. cruzi trypomastigotes (ratio 1 parasite/cell) for 22 h, in the presence of AB+ adult human or autologous cord plasma. The proportion of infected monocytes (A) and the number of amastigotes per infected cell (B) were determined by microscopic examination. The parasitic index (C) evaluates the global parasite burden (see M&M). Results are expressed as mean ± SEM (n = 10 in each group). *: p < 0.05, **: p < 0.05, ***: p < 0.005 as compared to M+B- monocytes cultures with AB+ adult plasma (Mann–Whitney–Wilcoxon test).

Figure 3

Intracellular amastigote multiplication in monocytes from M+B- and M-B-newborns, infected in vitro with T. cruzi trypomastigotes (ratio 1 parasite/cell) in the presence of AB+ adult human plasma or autologous cord plasma. The number of amastigotes per infected cell was determined by microscopic examination at 22 and 72 h. Results are expressed as mean ± SEM (n= between 6 and 12). *: p < 0.05, as compared to monocytes cultured with AB+ adult plasma (Mann–Whitney–Wilcoxon test).

Figure 4

Parasite burden of monocytes from M-B- and M+B- cord blood infected with low doses of T. cruzi trypomastigotes and cultured during 96 h in the presence of autologous cord plasma. After 96 h, cultures were stopped, cells were colored, and we counted the number of infected cells and of intracellular amastigotes per infected cell. The index corresponded to the product of both parameters and evaluates the global parasite burden. Results are expressed as mean ± SEM (n = 5). *: p < 0.05 compared with M-B- monocytes cultured with the same parasite ratio (Mann–Whitney–Wilcoxon test).

Figure 5

T. cruzi-specific Ab in the plasma of M+B- newborns before and after Ab depletion by immunoadsorption on adsorbent containing a T. cruzi antigenic extract or a mock IAS (Sepharose saturated with glycine). Ab were measured by ELISA. Sera from adults infected or not with T. cruzi were used as positive and negative controls.

Figure 6

Intracellular amastigotes in monocytes from M+B- newborns after 96 h of infection. Monocytes were infected with T. cruzi trypomastigotes at a ratio of 1 parasite/20 cell in the presence of autologous M+B- cord plasma depleted or not from T. cruzi-specific Abs: X axis: “+”: Abs are present (undepleted plasma); “±”: part of Abs are depleted (cf. Figure 5); “−“Abs are totally depleted. The number of amastigotes per infected cell was determined by microscopic examination. Results are expressed as mean ± SEM (n = 5). *: p < 0.05, as compared to the presence of Abs, #: p < 0.05 as compared to half-depleted Abs (Mann–Whitney–Wilcoxon test).