HIV-1 Nef breaches placental barrier in rat model

Abstract

The vertical transmission of HIV-1 from the mother to fetus is known, but the molecular mechanism regulating this transmission is not fully characterized. The fetus is highly protected by the placenta, which does not permit microbial pathogens to cross the placental barrier. In the present study, a rat model was established to observe the effect of HIV-1 protein Nef on placental barrier. Evans blue dye was used to assay permeability of placental barrier and fourteen day pregnant Sprague Dawley rats were injected intravenously with 2% Evans blue dye along with various concentrations of recombinant Nef. After an hour, animals were sacrificed and dye migration was observed through the assimilation of peripheral blood into fetus. Interestingly, traces of recombinant Nef protein were detected in the embryo as well as amniotic fluid and amniotic membrane along with placenta and uterus. Our study indicates that recombinant HIV-1-Nef protein breaches the placental barrier and allows the migration of Evans blue dye to the growing fetus. Further the concentration of Nef protein in blood is directly proportional to the intensity of dye migration and to the amount of Nef protein detected in uterus, placenta, amniotic membrane, amniotic fluid and embryo. Based on this study, it can be concluded that the HIV-1 Nef protein has a direct effect on breaching of the placental barrier in the model we have established in this study. Our observations will be helpful to understand the molecular mechanisms related to this breach of placental barrier by Nef in humans and may be helpful to identify specific Nef inhibitors.

Figure 1. Evans blue dye migration in rat whole embryo after an hour of injecting recombinant Nef at different concentration.

A: Without injection, B: Only Dye, C: Dye+250 µg Nef, D: Dye +500 µg Nef.

Figure 2. Entrenched Evans blue dye in the amniotic membrane of rat fetus, after an hour of intra-venous injection with different concentrations of recombinant Nef.

A: Without injection, B: Only Dye, C: Dye+250 µg Nef, D: Dye+500 µg Nef.

Figure 3. Quantification of Evans blue dye (OD at 590) present (within an hour) in brain and different fetal tissue isolates from 14 day pregnant Sprague Dawley rats injected intravenously without and with recombinant Nef and ASK-1 protein.

(A) Brain (B) Uterus (C) Placenta (D) Amniotic membrane. Three different bars in each set represent 0, 250 and 500 µg of recombinant Nef and 500 µg of ASK-1 injected intravenously along with Evans blue dye in the experimental animals. As data represent ±SEM of 3 separate experiments in duplicate and changes were considered as significant at *p≤0.05, **p≤0.01 and ***p≤0.001.

Figure 4. Detection of intravenous recombinant Nef protein migrated (within an hour) in brain and different fetal tissue isolates from 14 day pregnant Sprague Dawley rats injected intravenously without and with recombinant Nef protein.

(A) Brain (B) Uterus, (C) Placenta, (D) Amniotic membrane, (E) Amniotic fluid and (F) fetus after breaching the placental barrier. Three different bars in each set represents 0 µg, 250 µg and 500 µg of recombinant Nef injected intravenously along with Evans Blue dye in these animals As data represent ±SEM of 3 separate experiments in duplicate and changes were considered as significant at *p≤0.05, **p≤0.01 and ***p≤0.001.

Figure 5. Quantification of migrated recombinant Nef (within an hour) in fetal tissues.

Uterus, Amniotic membrane, Amniotic fluid and Embryo of female rats injected with 250 µg and 500 µg of recombinant Nef protein intravenously. As data represent ±SEM of 3 separate experiments in duplicate and changes were considered as significant at *p≤0.05, **p≤0.01 and ***p≤0.001.

Figure 6. Localization of recombinant Nef protein (green) and E-cadherin (red) in different fetal tissues through immuno-fluorescence staining.

The panel consist of set of figures; uterus, placenta, amniotic membrane and embryo. Blue colour shows the nuclear staining with DAPI whereas overlapping images are merged for Nef and E-cadherin localization. The fetal tissues were isolated from rats after an hour of intra-venous injection with 500 µg of recombinant Nef protein.