Stimulation of HIV-1 replication in BeWo cells was completely abolished when DBL3γ-732 (5 μM) was preincubated with 4 μM Fab of monoclonal antibody A5.13 (P = 0.03; Fig. 1b). This anti-DBL3γ-732-specific monoclonal antibody inhibits CSA binding by P. falciparum-infected erythrocytes (manuscript in preparation). The Fab was used because BeWo cells express IgG Fc receptors, which are known to modulate HIV-1 replication. When used alone, Fab A5.13 had no effect on HIV-1 replication or on BeWo cell viability (not shown). This indicates that blocking of DBL3γ-732 binding to CSA by Fab A5.13 prevented the modulation of viral replication. Preincubating DBL3γ-732 with the Fab of monoclonal antibody E4-2, specific for an irrelevant P. falciparum antigen (F. Nato, unpublished), did not abolish stimulation of HIV-1 replication (not shown). Furthermore, Fab E4-2 alone had no effect on HIV-1-infected BeWo cells (not shown). DBL3γ-732 had no effect on HIV-1 replication when we used CD4-positive T lymphocytes, which do not express surface-exposed CSA, in place of BeWo cells (not shown).
As a result of its known role in HIV-1 replication in T cells and HIV-1-infected placenta cells , as well as in placental malaria infection  we quantified TNF-α production in the BeWo cell culture supernatants. There was a dose-dependent relationship between added DBL3γ-732 and the quantity of TNF-α released into the supernatants irrespective of whether the BeWo cells were infected (VSV-G) or not (Delta Env control). TNF-α in these culture supernatants rose from undetectable in the absence of DBL3γ-732 up to 250 pg/ml at the highest dose. This indicates that TNF-α induction is DBL3γ-732 dependent. Furthermore, this was DBL3γ-732 specific, because the supernatants from parallel cultures incubated with DBL1α-varO contained no detectable TNF-α (not shown). The addition of recombinant TNF-α (0.5, 5 and 50 ng/ml) to pseudotyped HIV-1-infected BeWo cells resulted in a dose-dependent increase of HIV-1 replication (not shown). We next quantified IL-8 in the supernatant of infected BeWo cells cultivated in the presence of escalating doses of DBL3γ-732 (Fig. 1a); no detectable IL-8 was found (not shown).
We have shown here a marked increase in viral replication in BeWo cells at high DBL3γ-732 doses and a moderate inhibition with low DBL3γ-732 doses. It is noteworthy that this biphasic response is mirrored in field studies, in which high placental malaria parasitaemia is associated with an increased risk of in-utero HIV-1 MTCT, whereas low placental malaria parasitaemia is associated with a lower risk of transmission . The demonstration that recombinant DBL3γ-732-induced TNF-α production in BeWo cells is, to our knowledge, the first evidence for the stimulation of a placental cell type for TNF-α production by a specific P. falciparum adhesin. Such a stimulation may contribute to the elevated TNF-α levels in the placenta of P. falciparum-infected women , thus generating a specific cytokine milieu that influences cellular immune response(s) within the placenta . Furthermore, TNF-α increases the expression of additional receptors involved in Plasmodium-infected erythrocyte binding such as interstitial cell adhesion molecule type 1 also involved in the adhesion of peripheral blood monocytes to the trophoblast cells . The DBL3γ-732-triggered TNF-α production in BeWo cells could stimulate HIV-1 replication, possibly via long-terminal repeat-mediated activation as reported in ACH-2 cells . Recombinant exogenous TNF-α also stimulated HIV-1 replication in BeWo cells. Although this study strongly implicates TNF-α, we cannot at this stage exclude other factors.
The authors thank Sévérine Loizon, Ines Vigan-Womas and Micheline Guillotte for communicating unpublished information, Ayman Khattab and Mo-Quen Klinkert for the DBL3γ-732 gene, and Farida Nato and Hélène Souchon for the gift of the mouse monoclonal antibodies A5.13 and E4-2.
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