Differential placental immune mediators in pregnant women with preeclampsia infected or not with SARS-CoV-2

Technical Summary: Differential Placental Immune Mediators in Pregnant Women with Preeclampsia Infected or Not with SARS-CoV-2

Problem Statement
Pregnancy involves a complex immunological adaptation to tolerate the semi-allogeneic fetus while maintaining defense against pathogens. SARS-CoV-2 infection during pregnancy and preeclampsia (PE) are both associated with adverse maternal and neonatal outcomes. While SARS-CoV-2 enters host cells via the ACE2 receptor, and the placenta is rich in ACE2, the specific immunological modulation at the maternal-fetal interface when these two conditions co-occur remains poorly understood. There is a critical gap in knowledge regarding how the placental immune response is altered in pregnant women with both COVID-19 and preeclampsia compared to those with either condition alone, particularly concerning the expression of immune regulatory molecules (e.g., HLA-G, PD-1/PD-L1, CD28/CTLA-4) and their impact on disease severity and fetal outcomes.

Methodology
This was a descriptive cross-sectional study involving 113 pregnant women (aged 18–44) recruited between July and December 2020 in Brazil. The cohort was stratified into three groups:

1. C-NPE (n=45): SARS-CoV-2 positive (confirmed by RT-PCR or serology), no preeclampsia.
2. NC-PE (n=44): Preeclampsia, SARS-CoV-2 negative.
3. C-PE (n=24): SARS-CoV-2 positive and preeclampsia.

Data Collection and Analysis:

• Systemic Level: Peripheral blood was collected pre-delivery. Circulating levels of 35 immunological mediators (cytokines, chemokines, growth factors) were quantified using a multiplex Luminex® assay.
• Placental Level: Placental fragments were collected immediately post-delivery.
  • Histology: Hematoxylin and eosin (H&E) staining was used to assess macroscopic and microscopic alterations (e.g., infarction, fibrin deposition, villous hypoplasia).
  • Viral Detection: Immunofluorescence and immunohistochemistry (IHC) were performed to detect the SARS-CoV-2 Spike (S) protein and the ACE2 receptor.
  • Immune Mediators: IHC was used to evaluate the expression and localization of regulatory molecules: HLA-G (pan-isoform and soluble HLA-G5), PD-1, PD-L1, CD28, and CTLA-4.
• Statistical Analysis: Data were analyzed using ANOVA or Kruskal-Wallis tests followed by post-hoc tests. Multinomial logistic regression was employed to identify independent associations between molecular expression and clinical outcomes.

Key Contributions and Results

• Systemic Immune Profiles:

  • Pregnant women with preeclampsia (both NC-PE and C-PE) exhibited significantly higher circulating levels of IL-12, HGF, and IL-4 compared to the C-NPE group.
  • The C-NPE group showed a distinct inflammatory profile with higher levels of IP-10 and IL-2.
  • Multivariate analysis confirmed that IL-4, IL-12, HGF, and IFN-α were independently associated with the preeclampsia groups.

• Placental Viral Presence and ACE2 Expression:

  • The SARS-CoV-2 Spike protein was detected in 22% of C-NPE placentas and 33% of C-PE placentas.
  • Spike protein expression correlated with disease severity in mothers and newborns and was associated with placental microscopic changes.
  • ACE2 expression was high in fetal cells across all groups but was significantly lower in the C-NPE group compared to the NC-PE group. Lower ACE2 levels in C-NPE were associated with unfavorable newborn outcomes.

• Immune Regulatory Molecules (Checkpoint Pathways):

  • PD-L1: Expression was significantly lower in the C-NPE and C-PE groups compared to the NC-PE group. Reduced PD-L1 on the maternal side of the placenta was associated with histological alterations and severe maternal disease.
  • PD-1: Expression was significantly higher in the C-PE group compared to both C-NPE and NC-PE groups. Increased PD-1 in the C-PE group was associated with severe newborn outcomes.
  • CD28/CTLA-4: The C-PE group showed increased CD28 and decreased CTLA-4 expression compared to other groups. High CD28 expression was associated with neonatal severity in the C-NPE group.
  • HLA-G: While total HLA-G expression was similar across groups, soluble HLA-G5 levels were lower in C-NPE than in NC-PE. However, elevated HLA-G5 was associated with histological changes in COVID-19 groups.

• Correlations and Models:

  • In the C-PE group, a complex reciprocal relationship was observed: a negative correlation between PD-1 and PD-L1, and a positive correlation between PD-1 and HLA-G.
  • The study suggests that in C-PE, the combination of reduced CTLA-4/PD-1 and increased CD28 may facilitate a cellular response against the virus, while increased HLA-G helps minimize cytotoxic effects on the placenta.

Significance and Claims
The authors claim that their findings provide a mechanistic explanation for the clinical observation that pregnant women with preeclampsia infected with SARS-CoV-2 may present with a less severe form of COVID-19 compared to those with COVID-19 alone.

The paper posits that immunological homeostasis at the placental interface is critical. Specifically:

1. The reduced expression of CTLA-4 and PD-1 in the C-PE group, coupled with increased CD28, may contribute to a more effective cellular immune response against the infectious agent.
2. The increased expression of placental HLA-G in this context may exert an inhibitory effect on immune cells, minimizing cytotoxic damage to the placenta and preventing fetal rejection.
3. The study highlights that the severity of maternal or fetal disease depends on which side of the placental interface exhibits reduced PD-L1 expression and the degree of local immune inhibition.

The authors conclude that these specific immunological adaptations in the placenta of women with both conditions help maintain pregnancy and fight the virus, offering a partial explanation for the divergent clinical outcomes observed between the study groups. The paper does not propose new therapeutic interventions but rather offers an explanatory model for the observed immune regulation.