Disturbances in maternal immune function are widely recognized as a mechanism underlying the pathogenesis of preeclampsia[14; 15]. There is growing interest in immune-based therapies for preeclampsia and the potential role of immune-related biomarkers in early prediction of preeclampsia during pregnancy. However, current research on the immune homeostasis and immune therapies for preeclampsia has primarily focused on decidua and placental tissues[16; 17]. In this study, we performed an analysis of blood RNA-seq data to explore biomarkers that are highly correlated with preeclampsia. We experimentally validated these biomarkers and preliminarily assessed their predictive value. Additionally, we used gene set enrichment analysis (GSEA) to predict their potential biological functions and used single-cell RNA-seq to predict the expression of selected genes in maternal circulating immune cells. Furthermore, we conducted cell communication and ligand-receptor analysis to explore potential abnormal cell communication processes in the maternal circulation during the pathogenesis of preeclampsia, providing new insights for the treatment of established preeclampsia.
By performing GO analysis on differentially expressed genes, we discovered that immune dysfunction and immune environment abnormalities play important roles in the pathogenesis of preeclampsia, both in the placenta and the blood system. Subsequently, using WGCNA analysis, we identified F2RL1 and GZMH as highly correlated biomarkers with early-onset preeclampsia. Since the samples in our analysis were collected in late pregnancy, we first validated the analysis results using maternal plasma samples from late pregnancy. To further assess the potential predictive value of these genes for preeclampsia, we conducted experiments using plasma samples from early pregnancy (approximately 11–13 weeks of gestation). The results indicated a significant increase in F2RL1 expression and a significant decrease in GZMH expression in the preeclampsia group. Combining the experimental results, binary logistic regression analysis revealed that both selected biomarkers have discriminative value for preeclampsia, and the combination of F2RL1 and GZMH achieves the optimal predictive performance. In summary, the above analysis highlights the significant role of immune environment abnormalities in preeclampsia and identifies F2RL1 and GZMH as biomarkers with high predictive value for preeclampsia.
F2RL1, also known as PAR2 (Protease-Activated Receptor 2), encodes a member of the G protein-coupled receptor 1 protein family[18; 19; 20]. Previous studies have suggested that activation of this receptor can stimulate smooth muscle relaxation, vasodilation, increased blood flow, and decreased blood pressure. This protein also plays an important role in inflammation[21; 22], innate and adaptive immunity, and often acts as a promoter of inflammatory responses. Existing research on F2RL1 in pregnancy or preeclampsia has mainly focused on placental tissue. Studies by Al-Ani B et al and Hirai C et al[23; 24]. have claimed that activation of F2RL1 can promote the secretion of sFLT-1 by endothelial cells. In 2013, Al-Ani B et al. proposed that resveratrol can inhibit this pathological process. The study by Yuping Wang et al. confirmed these findings and suggested that the upregulation of neutrophil F2RL1 expression may be a pregnancy-specific change rather than specific to preeclampsia[25]. Our study results indicate that F2RL1 is primarily localized in NK cells and platelets in the blood system and may be regulated through the Toll-like receptor signaling pathway, pattern recognition receptor signaling pathway, and oxidative stress-induced intrinsic apoptotic signaling pathway. It is noteworthy that our research suggests the involvement of F2RL1 in vesicle targeting regulation. Vesicle targeting is a highly regulated and precise process that plays a critical role in various cellular functions[26; 27; 28; 29]. Throughout the pathogenesis of preeclampsia, there are disruptions in the secretion function of syncytiotrophoblasts and various immune cells, which have garnered significant attention[30; 31; 32]. Our study suggests that F2RL1 is mainly localized in NK cells in the blood system and may be involved in the dysregulation of secretion function in preeclampsia. This provides new insights for further mechanistic studies in the future.
GZMH (Granzyme H) is an enzyme family protein and an important regulatory molecule in cytotoxic lymphocytes (CTLs) and natural killer cells. Studies have shown that the expression of GZMH is regulated by cytokines and immune regulatory molecules[33]. When the immune system is stimulated by infection, tumors, or other diseases, the expression levels of GZMH may be upregulated, thereby enhancing the cytotoxicity of immune cells against abnormal cells. Additionally, GZMH is involved in regulating the production and release of cytokines, thereby influencing the magnitude and direction of immune responses. However, there is currently limited research exploring the potential role of GZMH in the pathogenesis and diagnosis of preeclampsia[34]. This study proposes that GZMH has good predictive value for preeclampsia. Combining single-cell sequencing analysis and GSEA analysis, we suggest that GZMH is mainly localized in NK cells in the maternal circulatory system and participates in the regulation of multiple physiological activities of NK cells, which is consistent with previous understanding of GZMH. Furthermore, we found that GZMH may affect lymphocyte chemotaxis. Excessive inflammation in multiple organs is a recognized characteristic of preeclampsia. Inhibiting the movement of lymphocytes to tissues such as the placenta is an effective method to alleviate excessive inflammation in these tissues. Therefore, further investigation of the potential mechanisms of GZMH in lymphocyte chemotaxis is warranted.
Through the analysis of the proportion of blood cells in the hematopoietic system, we found that an increased number of T cells and a decreased quantity of HSC G-CSF were the most significant changes in preeclampsia. Current research indicates that the numbers and activity of Th1 cells (producing pro-inflammatory cytokines such as interferon-gamma) and Th17 cells (secreting pro-inflammatory cytokines such as interleukin-17) in peripheral blood are significantly increased in preeclampsia patients, while the numbers and functions of regulatory T cells (Treg cells) are suppressed[35; 36; 37]. This study focused on analyzing the impact and potential therapeutic value of collective changes in T cell populations in preeclampsia. Based on the research findings, the imbalances in the numbers of different T cell types during the development of preeclampsia are partly influenced by the decreased relative regulatory strengths of various cell factors such as MIF, LCK, BTLA on T cells, and the significantly increased relative regulatory strengths of factors such as SELPLG, CD22, ADGRE5, and MK. Analysis of T cell secretion activities during the pathogenesis of preeclampsia revealed a significant increase in the intensity of CLEC, CD45, ITGB2, SELL, and PARs, while the secretion levels of factors such as MIF, CD99, ANNEXIN, IL16, LCK, and FLT3 were significantly decreased. Aberrant secretion by T cells is involved in the regulation of various immune modulation activities, including innate immunity, adaptive immunity, and immune responses. Based on the above analysis, we further explored potential approaches to block these abnormal regulatory processes. Targeted drugs can be broadly classified into four categories. The first category includes acetylsalicylic acid (C9H8O4) and salicylic acid (C9H8O3), with acetylsalicylic acid, also known as aspirin, widely used in the treatment of preeclampsia[38]. The second category includes drugs that affect the nervous system, and according to guidelines, antidepressant drugs are primarily used as secondary interventions for treating depression during pregnancy. The third category includes citric acid. Currently, there is no direct research linking citric acid to preeclampsia. However, the analysis results may suggest that a high dietary intake of citric acid may have certain preventive and therapeutic effects on preeclampsia. The fourth category includes tyrosine kinase inhibitors associated with FLT3. Both FLT1 and FLT3 are members of the receptor tyrosine kinase (RTK) family. FLT1 plays an important role in angiogenesis and vascular function regulation and has been extensively studied in the pathogenesis and diagnosis of preeclampsia. Research on FLT3 is mainly focused on the tumor field, especially in diseases related to the hematopoietic system[39; 40; 41]. Currently, there is a lack of research on the role of FLT3 in the pathogenesis of preeclampsia. Furthermore, tyrosine kinase inhibitors may serve as potential targets for preeclampsia treatment, but there is a lack of research on the efficacy and safety of such drugs for preeclampsia treatment.
This study still has certain limitations. On one hand, there is still insufficient research on single-cell transcriptomics of blood samples in preeclampsia, resulting in a lack of available datasets. In this study, quantitative analysis of the results and preliminary exploration of gene diagnostic performance were conducted using clinical samples only, without in-depth mechanistic research. Therefore, further experimental validation is needed to verify the results of single-cell transcriptomics analysis and predictions of protein-drug targets.