Coxsackievirus B4 Transplacental Infection Severely Disturbs Central Tolerogenic Mechanisms in the Fetal Thymus

Figures

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  Figure 1. Schematic representation of the experimental schedule adopted to explore the effect of in utero CV-B4 infection on transcription factors and autoantigens expression. Four mice females per male were caged together until successful fertilization. The day of detection of the vaginal plug is considered as the first day of gestation (day 1G). Pregnant mice were orally inoculated with CV-B4 E2, randomly, either at gestational days 10 (10G, 9 dams) or 17 (17G, 6 dams). Nine naive pregnant dams served as mock-infected negative controls. Offspring’s thymuses were harvested at day 17G, day 1, and day 5 from birth when mice were Mock- or CV-B4-inoculated at day 10G, and at day 1 and 5 from birth when mice were CV-B4-inoculated at day 17G. For each experimental condition (Mock- or CV-B4-inoculated at day 10G or 17G) and each sampling point (day 17G and days 1 and 5 from birth), thymuses were sampled from three litters of mice.

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  Figure 2. CV-B4 RNA quantification and detection in whole thymus and enriched TECs. (a). Viral RNA load in the whole thymus of offspring from CV-B4 E2-inoculated dams. Thymuses collected at different time-points (n = 6–13) were submitted to RNA extraction and then to viral RNA quantification by RT-qPCR. Results are expressed as copy numbers/100 ng of total RNA and represented as means ± SD. CV-B4 10G (▼): thymus harvested from mice born to dams inoculated with CV-B4 at Day 10 of gestation; CV-B4 17G (♦): thymus harvested from mice born to dams inoculated with CV-B4 at Day 17 of gestation. (b). Gel electrophoresis for CV-B4 RNA detection in enriched TECs by semi-nested PCR. (c). Gel electrophoresis for Car transcripts detection in enriched TECs, thymocytes and heart by PCR. M: Molecular weight ladder (100 bp); TECs (CV-B4): Enriched TECs harvested from in utero infected offspring; TECs (Mock): Enriched TECs harvested from age-matched controls; S: Sample; T+: Positive control (CV-B4 RNA); T−: Negative control (water PCR grade).

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  Figure 3. Transcription factors and autoantigens transcripts in the whole thymus. Complementary DNA obtained from mock- and CV-B4-infected thymuses (n = 5–7), harvested at different time-points, was submitted to quantification of Aire, Fezf2, Igf2, and Myo7 transcripts by qPCR. Transcripts levels were normalized to those of Oaz1. Relative gene expression in CV-B4 E2- vs. mock-infected thymuses was calculated, as described in Section 2, and expressed as 100 × mean 2−ΔΔCt values ± SD. The unpaired t-test was used for statistical analysis. ** p < 0.01. Mock (●) mock-infected thymuses harvested at different time points, from fetuses at day 17 of gestation (day 17G), and newborns at day 1 and day 5 from birth. CV-B4 10G (▼): thymus harvested from mice born to dams inoculated with CV-B4 at day 10 of gestation; CV-B4 17G (♦): thymus harvested from mice born to dams inoculated with CV-B4 at day 17 of gestation.

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  Figure 4. Transcription factors and autoantigens transcripts in enriched thymic epithelial cells. Whole thymuses were harvested, at different time-points, from offspring born to mock- and CV-B4-infected dams. Thymuses from fetuses or neonates of the same litter (n = 5–12) were pooled and subjected to TECs isolation. Transcripts levels for Aire, Fezf2, Igf2, and Myo7, in enriched TECs, were determined by RT-qPCR and normalized to those of Oaz1. Relative gene expression in TECs from CV-B4 E2- vs. mock-infected thymuses was calculated, as described in Section 2, and expressed as 100 × mean 2−ΔΔCt values ± SD. The unpaired t-test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. Mock (●) TECs from mock-infected thymuses harvested at different time points, from fetuses at day 17 of gestation (day 17G), and newborns at day 1 and day 5 from birth. CV-B4 10G (▼): TECs from thymus harvested from mice born to dams inoculated with CV-B4 at day 10 of gestation; CV-B4 17G (♦): thymus harvested from mice born to dams inoculated with CV-B4 at day 17 of gestation.

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  Figure 5. Transcription factors and autoantigens protein expression in the whole thymus. (a) Representative blots depicting the expression levels of AIRE, FEZF2, IGF2, and MYO7 proteins in CV-B4- and mock-infected thymus sampled at day 1. (b) Relative expression of AIRE, FEZF2, IGF2, and MYO7 determined by Western blot analysis. Proteins obtained from mock- and CV-B4-infected thymuses (n = 3–5), harvested at different time-points, were submitted to AIRE, FEZF2, IGF2, and MYO7 quantification by Western-blot analysis. Proteins levels were normalized to those of VINCULIN used as a loading control. Relative protein levels in CV-B4 E2- vs. mock-infected thymuses are represented as mean ± SD. The unpaired t-test was used for statistical analysis. * p < 0.05. Mock (●): mock-infected thymuses harvested at different time points, from fetuses at day 17 of gestation (day 17G), and newborns at day 1 and day 5 from birth. CV-B4 10G (▼): thymus harvested from mice born to dams inoculated with CV-B4 at day 10 of gestation; CV-B4 17G (♦): thymus harvested from mice born to dams inoculated with CV-B4 at day 17 of gestation.

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  Figure 6. Relationship between viral load and transcription factors, Aire (a) and Fezf2 (b), and autoantigens transcripts, Igf2 (c) and Myo7 (d), in the thymus. For each harvested thymus (CV-B4-infected or age-matched control), transcription factors and autoantigens transcripts together with the corresponding viral load were measured and plotted in the same graph. To better assess the effect of infection on transcription factors and autoantigens expression, an eventual correlation between both parameters was evaluated using the Spearmen’s correlation test. r is the Spearman’s correlation coefficient. p-values were corrected according to the Benjamini–Hochberg procedure (PBH). Thymuses harvested at day 17G (Day 17G (10G) (●)), day 1 (Day 1 (10G) (▲)) or day 5 (Day 5 (10G) (♦)) from mice born to dams inoculated (or mock) with CV-B4 at day 10 of gestation; thymuses harvested at day 1 (Day 1 (17G) (▼)) or day 5 (Day 5 (17G) (■)) from mice born to dams inoculated (or mock) with CV-B4 at day 17 of gestation. x and y axis were log-scaled.