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Role of the Maternal Gut Microbiota in Immune Activation at the Maternal-Foetal Interface: Impact on Preeclampsia and Offspring's Immune Development

Periodic Reporting for period 1 - DaPhNIs (Role of the Maternal Gut Microbiota in Immune Activation at the Maternal-Foetal Interface: Impact on Preeclampsia and Offspring's Immune Development)

Reporting period: 2019-07-16 to 2021-07-15

The main goal of the DaPhNIs project is to investigate how changes in gut microbiota during pregnancy impact immune responses at the maternal-foetal interface (MFI), and to determine the consequences on pregnancy outcomes as well as development of offspring’s immunity. Gut microbiota is an ecological community of commensal, symbiotic and pathogenic microorganisms, with a fundamental role in the maintenance of the host immune homeostasis. Accordingly, changes in the variety and composition of the host gut microbiota - a phenomenon termed “dysbiosis” - affect systemic immune responses and can disrupt the balance between pro- and anti-inflammatory activation leading to pregnancy-related diseases such as preeclampsia. In the DaPhNIs project we aim to investigate the immunological and molecular mechanisms through which changes in gut microbiota impact pregnancy outcome and fetal growth. Translational relevance of findings specifically to preeclampsia will be evaluated in a large cohort of patients, with implications potentially arising from the identification of novel biomarkers and of molecular mechanisms amenable to pharmacologic intervention.
To test this hypothesis, three different aims were pursued:
1. Determine how maternal dysbiosis affects phenotype and activation of B cells at the MFI, and the consequences on pregnancy outcome in a C57/BL6 murine model.
2. Determine how maternal dysbiosis affects maternal-foetal transfer of cells, and the consequences on development and function of the neonatal immune system in a C57/BL6 murine model.
3. Identify novel human biomarkers for preeclampsia.
1.2.1 Work Package 1
Gut maternal dysbiosis, induced by vancomycin-treatment, increases fetal resorption, reduces Flt-1 secretion, and significantly impact placental efficiency by reducing placental:fetal ratio. Evidence of lower placental efficiency was confirmed by a reduction of the labyrinth area (Figure 1). Consistently, detection of altered expression of CD31 and alpha smooth muscle actin (alpha-SMA) in the placental tissue of vancomycin-treated dams, confirmed defective vascular development and spiral artery remodeling. Of note, we also detected higher hypoxia-inducible factor 1 alpha (Hif-1α) expression (Figure 2).
Our preliminary data indicate that maternal dysbiosis in pregnant dams, induced by antibiotic treatment during gestation, is associated with a marked increase of B cells at the MFI (ed14.5). To better assess the effect of vancomycin-treatment on placental B cells, we isolated placental B cells at ed14.5 characterized them and evaluated cytokine production after 5 hours culture with PMA/ionomycin. Placental B cell characterization showed a significant up-regulation of follicular type II (FO-II) B cells in vancomycin-treated dams and a significant higher production of IL-10 compared to control (Figure 3).
During pregnancy, uterine NK (uNK) cells make up the vast majority of the maternal leukocytes, constituting 70% of the lymphocyte fraction. In our in vivo model we assessed that placental NK cells isolated from vancomycin-treated dams had a significant reduction in IFN-γ secretion and NKG2D expression compared to controls. Of note, placental NK cells of treated dams showed also increased TNF-α production. Accordingly, we found that vancomycin-treatment significantly reduced the number of placental DBA+ NK cells and VEGF-C expression in placental tissue (Figure 4).
Finally, to address whether the increased B cell population could be responsible for NK cell immune modulation, we performed a co-culture experiment. Placental B cells from control and vancomycin treated dams were co-cultured for 72 hours with control placental NK cells in the presence of PMA/ionomycin. As shown in Figure 5, after 72 hours of co-culture we detected significant lower IFN- secretion in NK cells co-cultured with vancomycin-derived B cells compared to control, indicating a modulatory effect of B cells on NK cell immune function.
1.2.2 Work package 2
To determine the role of transferred maternal cells in the development of the offspring’s immune system. Pregnant dams were treated with oral vancomycin and sacrificed at ed14.5 fetal livers were collected, and maternal CD45.2+ cells isolated. Considering the very low number of maternal cells identified in the fetal liver we decided to characterize the immune fetal population. We observed a significant reduction of total immune cells which was mostly due to a reduction of CD3-CD4+ cells, and reduction of secondary lymphoid organ (SLO) development (Figure 6).
To determine whether maternal dysbiosis is associated with functional deficits in the offspring’s immune system and capacity to combat neonatal infections, pups were subjected to systemic infection with E. coli at ppd14. Of note, we did observe significant reduction of E. coli translocation in the spleen of pups born to vancomycin-treated compared to controls dams, and limited immune activation (Figure 7).
1.2.3 Work package 3
To identify novel biomarkers of PE, blood and stool samples were collected from pathological pregnant women and women at high (HR) and low risk (LR) of developing PE.
We detected significant lower alpha-diversity in the fecal microbiota of HR women compared to LR at the 1st trimester of pregnancy, and between pathological and LR women at the 2nd trimester, suggesting the possible presence of a cluster shift associated with disease onset (Figure 8). Characterization of the microbial composition also showed significant differences in the microbial distribution of the different groups, suggesting the presence of cluster shifts associated with PE. (Figure 9). Furthermore, we evaluated whether PE is associated with changes in maternal-fetal transfer of cells. Preliminary data show that, HR group has higher fetal microchimerism at the 1st trimester compared LR. These data suggest that monitoring of fetal microchimerism could be a useful tool to predict pregnancy outcome (Figure 10).
Dissemination: the project results were presented twice at the World of Microbiome Conference (2019 and 2020), at the Humanitas Research Day and Departmental seminar session. In 2019 the ER participate to the Meet Me Tonight event, where she could foster scientific collaborations and disseminate the project to high school and elementary students. Finally, during the past 2 years the ER kept an active communication though her professional portals such as LinkedIn, ResearchGate and the MCAA website.
Exploitation: The results gained during the DaPhNIs project will enable the identification of dysregulated biomarkers and the development of in vitro diagnostic tools which will lead to more efficient clinically targeted prevention, stratification and intervention.
The identification of multiomic biomarkers involved in PE pathogenesis, which can be detected before disease onset, will enable the development of in vitro diagnostic tests improving early diagnosis and facilitating prediction of maternal and fetal outcomes. Better classification of PE population will lead to more efficient clinically targeted intervention (eliminating unnecessary testing and surveillance, and improving targeted intensive management), prevention of PE (reduction of maternal long term healthy-related issues), reduction of neonatal small gestation and early delivery (reduction of neonatal long term healthy-related issues).
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