Application of immune costimulatory factor TNFSF9 / TNFRSF9 as marker in preparation of preeclampsia intervention product

Abstract

The invention discloses an application of an immune costimulatory factor TNFSF9 / TNFRSF9 as a marker in preparation of a preeclampsia intervention product, and the immune costimulatory factor as an intervention target comprises TNFSF9 and / or TNFRSF9. According to the application, activation and immune response of immune cells of the placenta are accurately regulated and controlled by targeting a TNFSF9 / TNFRSF9 signal channel (signal axis), so that immune system disorder is effectively improved, local and systemic inflammatory response is relieved, the immune homeostasis of the placenta is recovered, and the pathophysiological process of preeclampsia is relieved.

Description

Technical Field

[0001] This application relates to the field of clinical medical technology, and more specifically, to the application of the immune co-stimulatory factor TNFSF9 / TNFRSF9 as a biomarker in the preparation of preeclampsia intervention products. Background Technology

[0002] Preeclampsia (PE) is a unique condition that occurs only during pregnancy, typically first appearing after 20 weeks of gestation. It is clinically characterized by hypertension and proteinuria, and is often accompanied by placental dysfunction, intrauterine growth restriction (IUGR), and multiple organ system dysfunction (such as kidney, liver, and central nervous system abnormalities). PE is a leading cause of maternal morbidity and mortality worldwide. In severe cases, it can progress to life-threatening complications such as eclampsia, HELLP syndrome (named for its three main characteristics: hemolysis, elevated liver enzymes, and low platelets), or placental abruption. Its complex pathological process and unpredictable progression pose significant challenges to clinical management.

[0003] Despite a deepening understanding of PE in clinical practice, effective interventions to cure the disease remain lacking. Treatment primarily focuses on symptom relief and delaying delivery to improve fetal outcomes. Commonly used medications include antihypertensive drugs such as methyldopa and labetalol, as well as magnesium sulfate for preventing seizures. Additionally, glucocorticoids may be used to improve placental growth retardation or promote fetal lung maturation. However, these medications are mostly supportive or palliative treatments and cannot fundamentally address the core mechanisms of the disease, particularly the pathological changes caused by immune activation or placental perfusion disorders. While immunosuppressants such as glucocorticoids can suppress some inflammatory responses, long-term use has significant side effects and cannot achieve precise localization and regulation of lesions. Therefore, there is an urgent need to explore novel treatment strategies with strong targeting, clear mechanisms, and clinical translational potential. Summary of the Invention

[0004] This application addresses the shortcomings of existing methods by proposing the application of immune co-stimulatory factors TNFSF9 / TNFRSF9 as biomarkers in the preparation of preeclampsia intervention products, in order to solve the technical problems of lack of targeting, unclear intervention mechanism, and difficulty in clinical translation of related technologies.

[0005] The application of immune co-stimulatory factors TNFSF9 / TNFRSF9 as biomarkers in the preparation of preeclampsia intervention products, wherein the immune co-stimulatory factors as intervention targets include TNFSF9 and / or TNFRSF9.

[0006] Alternatively, the product can be used to intervene in preeclampsia before 20 weeks of gestation.

[0007] Alternatively, the product may be applied to the placenta.

[0008] Furthermore, the product is used to block the signaling axis of the immune co-stimulatory factor TNFSF9 / TNFRSF9.

[0009] Alternatively, the product is used to inhibit or silence the TNFSF9 gene and / or the TNFRSF9 gene, and / or to act by blocking TNFSF9 / TNFRSF9 signaling.

[0010] Alternatively, the product can be used to reduce the activation of immune cells in the placenta.

[0011] Furthermore, the immune cells include macrophages and / or natural killer cells.

[0012] Alternatively, the product can be used to alleviate elevated systolic blood pressure and proteinuria that occur during pregnancy.

[0013] Alternatively, the product may be used to improve placental and / or fetal development.

[0014] The beneficial technical effects of the technical solutions provided in this application include: This application targets the TNFSF9 / TNFRSF9 signaling pathway (signaling axis) to precisely regulate the activation of placental immune cells and immune responses, thereby effectively improving immune system disorders, reducing local and systemic inflammatory responses, restoring placental immune homeostasis, and thus alleviating the pathophysiological process of preeclampsia.

[0015] This application targets the TNFSF9 / TNFRSF9 signaling pathway, which helps restore placental immune homeostasis. By precisely regulating the immune response, it reduces the immune system's attack on the placenta, improves placental blood flow and function, and provides a healthier developmental environment for the fetus.

[0016] This application aims to improve the pathophysiological process of preeclampsia by regulating the function of immune cells, alleviating clinical symptoms such as hypertension and proteinuria, delaying or preventing disease progression, and reducing the risk of pregnancy complications.

[0017] Additional aspects and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0018] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 To analyze immune cell infiltration in mouse placental transcriptome data after intervention with Fusobacterium nucleatum using the CIBERSORTx deconvolution algorithm; Figure 2 To analyze the effect of inhibiting TNFSF9 or TNFRSF9 expression in human trophoblast cells using shRNA on trophoblast cell proliferation; Figure 3 To analyze the effect of inhibiting TNFSF9 or TNFRSF9 expression in human trophoblast cells using shRNA on the invasive ability of trophoblast cells; Figure 4 To analyze the effect of inhibiting TNFSF9 or TNFRSF9 expression in human trophoblast cells using shRNA on trophoblast cell apoptosis; Figure 5 The results of clinical manifestations in mice after specific knockout of Tnfsf9 or Tnfrsf9 in the placenta show changes in blood pressure and urinary protein. Figure 6 To investigate the development of the mouse placenta and fetus after specific knockout of Tnfsf9 or Tnfrsf9 in the mouse placenta; Figure 7 To verify the pathological changes in the placenta after specific knockout of Tnfsf9 or Tnfrsf9 in mouse placenta, hematoxylin-eosin stained mouse placental sections were used. Figure 8 To verify the immune cell infiltration in mice after specific knockout of Tnfsf9 or Tnfrsf9 in the placenta, CD45 immune cells stained with immunofluorescence (red area in the figure) were used. Figure 9 The change in the proportion of M1 macrophages in mouse placenta after specific knockout of Tnfsf9 or Tnfrsf9 in mouse placenta; Figure 10 This study investigated the changes in the proportion of CD107a-positive and IFN-γ-positive NK cells in mouse placenta after specific knockout of Tnfsf9 or Tnfrsf9 in mouse placenta. Detailed Implementation

[0019] The embodiments of this application are described below with reference to the accompanying drawings. It should be understood that the embodiments described below with reference to the accompanying drawings are exemplary descriptions for explaining the technical solutions of the embodiments of this application, and do not constitute a limitation on the technical solutions of the embodiments of this application.

[0020] Those skilled in the art will understand that, unless specifically stated otherwise, the terms "described" and "the" as used herein may also include plural forms. It should be further understood that the term "comprising" as used in the specification of this application means the presence of the stated features, integers, steps, operations, elements, and / or components, but does not exclude other features, information, data, steps, operations, elements, components, and / or combinations thereof supported by the art. The term "and / or" as used herein refers to at least one of the items defined by the term; for example, "A and / or B" can be implemented as "A," or as "B," or as "A and B."

[0021] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0022] In recent years, with the development of high-throughput sequencing and multi-omics technologies, the etiological mechanisms of preeclampsia have been continuously revealed. Among them, infectious factors, especially Fusobacterium nucleatum (…), are… Fusobacterium nucleatum Infection with *Fusobacterium nucleatum* has been identified as playing a crucial role in the development of preeclampsia (PE). *Fusobacterium nucleatum* is an anaerobic, Gram-negative bacillus widely distributed in the mucosal microecology of the oral cavity and reproductive tract, and has previously been considered closely related to periodontitis, colorectal cancer, and bacterial vaginosis. During pregnancy, this bacterium can enter the uterus via the bloodstream and colonize placental tissue, disrupting local immune homeostasis and inducing placental structural and functional abnormalities.

[0023] At the molecular level, *Fusobacterium nucleatum* can significantly upregulate the expression of TNFSF9 (4-1BBL) and its receptor TNFRSF9 (4-1BB) in trophoblast cells, activating a typical immune co-stimulatory signaling pathway. This signaling axis plays a core regulatory role in immune responses such as enhanced natural killer cell (NK cell) function and macrophage polarization towards the M1 type. In an infected environment, its sustained activation leads to abnormal aggregation of immune cells and a persistent inflammatory response, causing imbalance in the placental immune microenvironment, impaired angiogenesis, increased apoptosis, and ultimately resulting in inadequate placental perfusion and exacerbation of PE symptoms.

[0024] The TNFSF9 / TNFRSF9 signaling pathway has been extensively studied in the field of tumor immunology, playing a crucial role in tumor vaccines, CAR-T therapy, and immune checkpoint inhibitor research. However, in the unique immune regulation of pregnancy, this signaling axis may be involved in the regulation of immune cell function at the maternal-fetal immune interface, representing a key point in the disruption of immune tolerance during pregnancy. Therefore, targeting the TNFSF9 / TNFRSF9 signaling pathway, starting from the pathological chain of infection triggering—immune activation—signal pathway abnormality, holds promise as a novel strategy for regulating placental immune homeostasis and alleviating preeclampsia.

[0025] Fusobacterium nucleatum infection is a significant pathogenic factor for preeclampsia. This pathogen can stimulate placental trophoblast cells, inducing abnormal expression of TNFSF9 and TNFRSF9, immune co-stimulatory molecules. This activates inflammatory polarization (M1 polarization, a pro-inflammatory phenotype) of macrophages at the maternal-fetal interface and increases cytotoxicity of NK cells, thereby triggering an inflammatory response that damages placental development and ultimately induces preeclampsia. Transcriptomic analysis shows a significant positive correlation between the expression levels of TNFSF9 and TNFRSF9 in placental tissue and the risk of preeclampsia. However, it remains unclear whether targeting and inhibiting the expression of TNFSF9 or TNFRSF9 in trophoblast cells can alleviate the pathological progression of preeclampsia.

[0026] To systematically verify the above hypotheses, a pathogenic model of preeclampsia was first constructed: Fusobacterium nucleatum was injected into pregnant mice to simulate pathogen-induced placental immune activation observed clinically. (Reference) Figure 1 Fusobacterium nucleatum significantly activated trophoblast cells, induced upregulation of TNFSF9 / TNFRSF9 expression, and was accompanied by the enrichment of M1 macrophages and NK cells. Further analysis of placental transcriptome data using the CIBERSORTx deconvolution algorithm revealed a significant increase in the number of M1 macrophages and NK cells in the placenta of infected mice, suggesting that the TNFSF9 / TNFRSF9 signaling axis may be involved in the pro-inflammatory activation of immune cells, thereby mediating the pathogenesis of preeclampsia.

[0027] CIBERSORTx is a tool based on deconvolution algorithms used to quantitatively infer cell type proportions from mixed RNA samples (such as BuIK RNAseq data). Its core principles include: a deconvolution model—constructing a cell type-specific gene expression feature matrix (signature matrix) from single-cell RNA-seq data, and performing linear deconvolution using support vector machine regression (SVR). The algorithm establishes a regression model by adjusting different nu values ​​(e.g., 0.25, 0.5, 0.75), calculating the cell type weight vector, and ultimately estimating the proportion of each cell type in the mixed sample. The mathematical foundation is based on the linear regression model Y = x·W + é, where Y is the gene expression matrix of the Bulk sample, x is the single-cell feature matrix, W is the cell proportion weight vector to be solved, and c is the residual. The weights are optimized by minimizing the mean squared error (MSE) between the predicted and actual values.

[0028] In in vitro experiments, shRNA technology was used to knock down the expression of TNFSF9 or TNFRSF9 in human placental trophoblast cells. The results showed that inhibition of either drug alone significantly improved the placental dysfunction phenotype. Specifically, it was able to rescue the reduced trophoblast proliferation induced by *Fusobacterium nucleatum*. Figure 2 The Transwell assay (which uses matrix gel to simulate the basement membrane barrier and assesses cell invasiveness by detecting the ability of cells to cross the matrix gel) demonstrated that it can salvage the impaired invasive ability of trophoblast cells. Figure 3 ) and an increase in placental cells that control apoptosis ( Figure 4 The significant reduction in apoptotic or necrotic cells represented by the Q1 and Q2 quadrants in the experimental group further supports the key role of this signaling axis in the pathological changes of trophoblast cells.

[0029] The purpose of shRNA technology is to achieve gene silencing. shRNA is introduced into cells via vectors (such as plasmids or viruses), cleaved into siRNA by the Dicer enzyme, and then binds to the RNA-induced silencing complex (RISC), specifically degrading target mRNA to achieve gene knockdown (typically with an efficiency of 70%-90%). This application utilizes shRNA technology for target confirmation: knocking down potential drug target genes (such as kinases and receptors), observing whether cell phenotypes are reversed after drug treatment (such as inhibition of proliferation and reduction of apoptosis), and verifying the effectiveness of the target.

[0030] In in vivo experiments, a trophoblast cell-specific gene knockout mouse model driven by the Cyp19a1-cre system was constructed. The Cyp19a1-cre system is a genetic tool that utilizes the Cyp19a1 (aromatase gene) promoter to drive the specific expression of Cre recombinase in estrogen-active tissues, and combines with the loxP site to achieve conditional gene knockout. By precisely targeting estrogen synthesis and signaling pathways, the Cyp19a1-cre system has become a key tool in research on reproductive health, oncology, metabolism, and neurological diseases.

[0031] refer to Figure 5 A, Cyp19a1 is expressed in placental trophoblast cells from day 5.5 of mouse gestation and drives the specific expression of Cre enzymes. This is achieved by using Tnfsf9... flox / flox Or Tnfrsf9 flox / flox Mice were mated with Cyp19a1-cre mice to obtain Tnfsf9. flox / flox;Cyp19a1-cre / cre With Tnfrsf9 flox / flox;Cyp19a1-cre / cre Genotype mice. Laboratory mice were housed under specific pathogen-free (SPF) conditions at a constant temperature of 20–25°C with a 12-hour day-night cycle. At 7.5 days of gestation, mice were injected via tail vein with *Fusobacterium nucleatum* (10...).4 (CFU / mL, 100 µL), and placental and embryonic tissues were collected on day 17.5 for analysis.

[0032] The results of the various analyses are as follows: Figure 5 The study aimed to specifically knock out Tnfsf9 or Tnfrsf9 in the mouse placenta, thereby improving elevated blood pressure and urinary protein levels induced by Fusobacterium nucleatum infection, suggesting an improvement in preeclampsia-like symptoms. (A) is a schematic diagram of the mouse experiment. (B) shows the blood pressure changes between the Tnfsf9 and Tnfrsf9 knockout groups and the control group; by day 17.5 of gestation, the blood pressure of the Tnfsf9 or Tnfrsf9 knockout group mice was significantly lower than that of the control group mice. (C) shows the urinary protein / creatinine ratio in mice at day 17.5 of gestation; the urinary protein / creatinine ratio of the Tnfsf9 or Tnfrsf9 knockout group mice was significantly lower than that of the control group mice.

[0033] Figure 6 This study aimed to specifically knock out Tnfsf9 or Tnfrsf9 in the mouse placenta to improve placental and fetal developmental damage induced by *Fusobacterium nucleatum* infection. (A) Placental weight at 17.5 days of gestation in mice was significantly higher in the Tnfsf9 or Tnfrsf9 knockout group than in the control group. (B) Fetal weight at 17.5 days of gestation in mice was significantly higher in the Tnfsf9 or Tnfrsf9 knockout group than in the control group. (C) Embryo resorption rate at 17.5 days of gestation in mice showed no difference between the Tnfsf9 or Tnfrsf9 knockout group and the control group.

[0034] Figure 7 To specifically knock out Tnfsf9 or Tnfrsf9 in mouse placenta and improve the pathological changes in placental occurrence induced by *Fusobacterium nucleatum*. The left side shows hematoxylin-eosin stained mouse placental sections, demonstrating that Tnfsf9 and Tnfrsf9 knockout improved *Fusobacterium nucleatum*-induced vascular stenosis and structural disorder in the labyrinthine region; the right side shows the statistical values ​​of these results.

[0035] Figure 8 To specifically knock out mouse placental Tnfsf9 or Tnfrsf9 to improve the infiltration of CD45-positive immune cells induced by Fusobacterium nucleatum. The left side shows immunofluorescence-stained CD45-positive immune cells (red areas in the image), and Tnfsf9 and Tnfrsf9 knockout reduced immune cell infiltration; the right side shows the statistical values ​​of these results.

[0036] Figure 9 Knocking out Tnfsf9 or Tnfrsf9 in mouse placenta reduced the increase in the proportion of placental M1 macrophages induced by Fusobacterium nucleatum infection, suggesting that the placental inflammatory environment was improved.

[0037] Figure 10 Knocking out Tnfsf9 or Tnfrsf9 in mouse placenta reduced the proportion of CD107a-positive and IFN-γ-positive NK cells induced by Fusobacterium nucleatum infection, suggesting that the placental inflammatory environment was improved.

[0038] The analysis results showed that, compared with the control group, pregnant mice in the Tnfsf9 or Tnfrsf9 gene knockout group had significantly reduced systolic blood pressure elevation and abnormal proteinuria in late pregnancy. Figure 5 B~C), placental and fetal weight recovery ( Figure 6 (A~C) indicates improved pregnancy outcomes. Histological analysis further showed that knockout of Tnfsf9 or Tnfrsf9 increased the ratio of the placental junction to the labyrinth region (A~C). Figure 7 ), promotes vascularization and reduces inflammatory cell infiltration ( Figure 8 In particular, knocking out Tnfsf9 significantly reduced the proportion of M1 macrophages in the placenta, while knocking out Tnfrsf9, although showing a decreasing trend, did not reach statistical significance. Figure 9 Furthermore, *Fusobacterium nucleatum* infection significantly increased the proportion of CD107a-positive and IFN-γ-positive NK cells in the placenta, suggesting that immune cells were in an inflammatory activated state; while this effect was significantly limited in Tnfrsf9-deficient placentas. Figure 10 This indicates that Tnfrsf9 plays an important regulatory role in NK cell activation.

[0039] The results above indicate that activation of the TNFSF9 / TNFRSF9 signaling axis mediated by *Fusobacterium nucleatum* infection may be one of the key molecular events in the development of preeclampsia. Research targeting this pathway for intervention not only has clear biological mechanistic support but also possesses significant clinical translational potential, providing a new research direction and theoretical foundation for developing mechanism-guided precision treatment strategies for preeclampsia.

[0040] Identifying and targeting the TNFSF9 / TNFRSF9 signaling pathway as a novel therapeutic target for preeclampsia (PE). Studies have shown that this pathway plays a crucial role in immune activation, inflammatory responses, and placental damage in PE. Functional validation using mouse models, trophoblast organoids, and primary cells demonstrates that the TNFSF9 / TNFRSF9 signaling axis is a core node in the immune regulation of PE. Based on this finding, it is further proposed that this pathway can be blocked using antibodies, RNA interference, or small molecule drugs to restore placental function and alleviate PE symptoms.

[0041] By blocking signaling pathways with antibodies, RNA interference, or small molecule drugs, a variety of therapies can be developed, including monoclonal antibodies, bispecific antibodies, ADCs, siRNA drugs, and small molecule kinase inhibitors. These therapies achieve effective treatment of the disease by precisely regulating signaling pathways. Combined with combination therapies, gene editing, cell therapy, and personalized treatment, these therapies promote the precision treatment of preeclampsia.

[0042] In summary, this application provides the use of the immune co-stimulatory factors TNFSF9 / TNFRSF9 as biomarkers in the preparation of preeclampsia intervention products, wherein the immune co-stimulatory factors, as intervention targets, include TNFSF9 and / or TNFRSF9. This application precisely regulates the activation of placental immune cells and immune responses by targeting the TNFSF9 / TNFRSF9 signaling pathway (signaling axis), thereby effectively improving immune system disorders, reducing local and systemic inflammatory responses, restoring placental immune homeostasis, and ultimately alleviating the pathophysiological process of preeclampsia.

[0043] Those skilled in the art will understand that the steps, measures, and solutions in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted. Furthermore, other steps, measures, and solutions in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and solutions in related technologies that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted.

[0044] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0045] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0046] The above description is only a partial implementation of this application. It should be noted that for those skilled in the art, other similar implementation methods based on the technical concept of this application, without departing from the technical concept of this application, also fall within the protection scope of the embodiments of this application.

Claims

1. The application of immune co-stimulatory factors TNFSF9 / TNFRSF9 as biomarkers in the preparation of preeclampsia intervention products, characterized in that, The immune co-stimulatory factors used as intervention targets include TNFSF9 and / or TNFRSF9.

2. The application as described in claim 1, characterized in that, The product is used to intervene in preeclampsia before 20 weeks of gestation.

3. The application as described in claim 1, characterized in that, The product acts on the placenta.

4. The application as described in claim 3, characterized in that, The product is used to block the signaling axis of the immune co-stimulatory factor TNFSF9 / TNFRSF9.

5. The application as described in claim 4, characterized in that, The product is used to inhibit or silence the TNFSF9 gene and / or the TNFRSF9 gene, and / or to act by blocking TNFSF9 / TNFRSF9 signaling.

6. The application as described in claim 4, characterized in that, The product is used to reduce the activation of immune cells in the placenta.

7. The application as described in claim 6, characterized in that, The immune cells include macrophages and / or natural killer cells.

8. The application as described in claim 1, characterized in that, The product is used to alleviate elevated systolic blood pressure and abnormal proteinuria that occur during pregnancy.

9. The application as described in claim 1, characterized in that, The product is used to improve placental and / or fetal development.

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