TOP2A drives T-cell infiltration and immune remodeling in cyclophosphamide-induced cystitis: a single-cell sequencing study with potential implications for interstitial cystitis
Minli Shi, Wantong Xue, Xiaodong Wen, Lei Pang
TL;DR
This study shows that TOP2A promotes T-cell infiltration in a rat model of bladder inflammation, suggesting it could be a new treatment target for interstitial cystitis.
Contribution
The study identifies TOP2A as a novel driver of T-cell infiltration and immune remodeling in cyclophosphamide-induced cystitis.
Findings
TOP2A is significantly upregulated in CYP-induced cystitis rat bladder tissues.
TOP2A correlates strongly with CD4+ and CD8+ T-cell infiltration in the model.
TOP2A is linked to oxidative phosphorylation and ribosomal pathways in bladder inflammation.
Abstract
To explore the potential mechanisms of interstitial cystitis (IC), we employed a cyclophosphamide (CYP)-induced cystitis rat model, a well-established tool for studying IC-like bladder inflammation and dysfunction. This study aimed to investigate the role of rhythmic genes and immune microenvironment remodeling in this model, focusing on TOP2A and its impact on T-cell infiltration. CYP-induced cystitis rat models were established using cyclophosphamide. Single-cell RNA sequencing was performed on bladder tissues to analyze cellular heterogeneity. Differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) identified rhythmic and immune-related gene clusters. TOP2A was validated via RT-PCR, Western blot, and immunohistochemistry (IHC). Statistical analyses assessed correlations between TOP2A, CD4 + T cells, and CD8 + T cells. Single-cell sequencing…
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Figure 8- —General Project of the Natural Science Foundation of Shanxi Province
- —2024 Clinical Scientific Research Project for Doctors of shanxi Provincial Medical Doctor Association
- —Scientific Research Project of Shanxi Provincial Health Commission
- —Open Project of the key Laboratory of Medical Molecular Virology, Ministry of Education/Health Commission of china.Fudan University
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Taxonomy
TopicsUrinary Bladder and Prostate Research · Bladder and Urothelial Cancer Treatments · Polyomavirus and related diseases
Introduction
Interstitial cystitis (IC) is a chronic urinary system disease with an unknown etiology. Clinically, it manifests as persistent pelvic pain, urinary urgency, and frequent urination, all of which severely impair patients’ quality of life [1]. The etiology and pathogenesis of IC remain incompletely understood [2–4], presenting significant challenges for diagnosis and treatment. Currently, there is a widespread consensus that its pathogenesis involves a complex interplay of multiple factors, such as dysfunction of the urothelial barrier, neurogenic inflammation, mast cell activation, and aberrant immune responses [5, 6]. Histopathological studies have revealed marked immune cell infiltration in the bladder tissues of IC patients, including T lymphocytes and B lymphocytes, along with upregulated expression of various inflammatory factors. These observations suggest that immune microenvironment dysregulation may play a pivotal role in the pathological progression of IC [7]. However, the specific cellular composition, functional states, and key molecular regulators that orchestrate this pathological immune microenvironment have yet to be fully elucidated.
The advent of single-cell RNA sequencing (scRNA-seq) technology has opened up a new avenue for exploring cell heterogeneity and the underlying molecular mechanisms of complex diseases, such as IC. In contrast to traditional bulk sequencing, this technology offers a higher level of resolution in dissecting cell heterogeneity and molecular mechanisms within complex diseases [8], thereby allowing us to more precisely identify disease-associated cell subpopulations and their gene expression profiles [9].
To delve deeper into the immunopathogenic mechanisms of IC, we employed an established rat model of cyclophosphamide (CYP)-induced cystitis. This CYP model exhibits several pathological characteristics observed in human IC, encompassing urothelial injury, mast cell activation, and marked infiltration of immune cells, notably T lymphocytes [10]. Despite the discrepancy between the acute nature of the CYP model and the chronic course of human IC, it still serves as a valuable and dependable tool for investigating immune-mediated processes in bladder inflammation.
In this study, we initially employed scRNA-seq technology to analyze the bladder cell landscape in CYP-induced cystitis, revealing a significant expansion of T lymphocytes in the disease group. To delve deeper into the underlying regulatory network, we conducted weighted gene co-expression network analysis for modular mining of differentially expressed genes, with a particular focus on the “immune module” and “rhythmic module,” both of which are highly correlated with the phenotype. The rhythmic module warrants special attention, as clinical observations have indicated that symptoms such as pain and urinary urgency in patients with IC often exhibit circadian fluctuations, frequently accompanied by sleep disturbances. This suggests a potential link between circadian rhythm regulation and disease progression [11]. Notably, intersection analysis of these two modules identified TOP2A as the sole overlapping hub gene. As a crucial topoisomerase involved in regulating DNA replication and cell cycle progression, TOP2A’s role in immune cell proliferation and activation has been documented in other inflammatory diseases; however, its function in the context of IC remains unclear.
Based on the above findings, the following objectives were established for this study: firstly, to verify that TOP2A is a key upregulated gene in the CYP-induced cystitis model; secondly, to investigate its functional relationship with T cell infiltration. We propose the hypothesis that TOP2A may participate in the remodeling of the local immune microenvironment of the bladder by regulating T cell dynamics. This study provides a potential new molecular basis for understanding the pathogenesis of CYP-induced cystitis, and offers a possible theoretical foundation for future research on potential therapeutic strategies targeting TOP2A in IC. The specific experimental scheme is shown in Fig. 1.
Fig. 1. Technology roadmap
Materials and methods
CYP-induced cystitis rat model construction and validation
Experimental animals
20 6–8-week-old female Sprague-Dawley rats were purchased from the Animal Experiment Center of Shanxi Provincial People’s Hospital. All experimental rats were kept in the SPF-grade pathogen-free animal facility of our center. The ambient temperature of the facility was 25 ℃, and 12 h of light and 12 h of darkness were alternated daily. Adequate care was given to the experimental animals to ensure that the experiments complied with the ethical requirements for animal experiments, and the protocols of the animal experiments were examined and approved by the Ethics Committee for Experimental Animals of Shanxi Provincial People’s Hospital [(2021) Provincial Medical Science Lun No. 154].
CYP-induced cystitis rat model construction
Based on previous experience and our prior study, this study established a CYP-induced rat model of interstitial cystitis. SD rats were randomly assigned to a control group and an experimental group according to a random number table, with 10 rats in each group. Rats in the experimental group were intraperitoneally injected with CYP (25 mg/kg) on days 1, 3, 5, 7, and 9, with continuous administration for 10 days; rats in the control group were intraperitoneally injected with an equal volume of DMSO/sterile saline mixture at the same time points. All rats were sacrificed 24 h after the last administration. During intraperitoneal injection, care was taken to aspirate the syringe to avoid introducing air into the intestines.
Success criteria for animal model construction
Von-Frey fiber palpation method
In accordance with the method described by previous researchers, individual rats were placed in stainless-steel mesh-bottomed cages enclosed by clear resin. The assessment of bladder pain was conducted on days 7 and 10 using Von Frey filaments. The lower abdomen (in the vicinity of the bladder) of the rats was stimulated in an up-down motion with calibrated Von Frey filaments of varying thicknesses (1 g, 2 g, 4 g, 8 g, 10 g, 15 g, 26 g). Positive behaviors included a sudden abdominal contraction and immediate licking and scratching of the pelvic skin following stimulation. A positive response to pelvic area stimulation was defined as follows: a sharp abdominal retraction was scored 1 point; jumping or immediate licking and touching after stimulation was scored 2 points; scratching the pelvic skin, as well as a sharp leg retraction or tongue-licking of the feet, was considered a positive response to plantar stimulation and scored 3 points. No response was assigned a score of 0 points. Each filament was tested 3 times, with a 5-second interval between each test. The lowest value of positive behavior was recorded as the mechanical pain threshold, and this process was carried out using a double-blind method.
Urodynamics
To assess bladder function, this study performed urodynamic measurements on rats under urethane anesthesia. After transurethral catheterization, physiological saline was infused at a constant rate of 10 mL/h, and at least three complete micturition cycles were recorded for each rat. The maximum bladder capacity was calculated using the formula “infusion time (min) × infusion rate (mL/min)”, and parameters such as maximum urethral pressure and voiding interval were extracted from the pressure dynamics data collected by the system, while the morphology of the urodynamic curves was also analyzed.
Bladder pathological manifestations
After completing the urodynamic measurements, three rats were randomly selected from both the experimental and control groups. These rats were sacrificed, after which the abdominal cavities were opened. The bladders were then excised, weighed to assess their general morphology, and dissected to observe the severity of edema and hemorrhage, thereby evaluating the degree of bladder inflammation. The bladder tissues were fixed in 4% paraformaldehyde for 24 h. Subsequently, they were routinely dehydrated using a graded alcohol series, made transparent with xylene, embedded in paraffin for sectioning, and sealed with HE-stained neutral resin. Finally, the histopathological changes in the bladder tissue structures of rats from the control and experimental groups were observed under a light microscope.
Single-cell sequencing analysis
Preparation of bladder single cell suspension
In this study, bladder tissues were collected from rats in each group. After mechanical disruption and sequential digestion with collagenase I, DNase I, and Accutase, a single-cell suspension was prepared. Subsequently, residual red blood cells were removed using red blood cell lysis buffer, and viable cells were enriched through centrifugation and filtration. The viability of the obtained cells was assessed using the Trypan blue staining method, confirming that the proportion of viable cells was over 80%. The final suspension was used for single-cell RNA sequencing analysis.
Single-cell RNA sequencing from CYP-induced cystitis rat bladder tissue
The nuclear DNA in the open chromatin region was digested by transposase. Subsequently, a 10×Genomics platform was utilized, which incorporated microfluidic technology and magnetic beads with Cell Barcodes. Cells were encapsulated into droplets during this process. The droplets containing cells were collected. Inside the droplets, cell lysis occurred, and magnetic beads captured mRNA. Under the action of reverse transcriptase, mRNAs were reverse-transcribed into cRNAs with Barcodes and UMIs cDNA. After incubation, the Gel Bead-in-Emulsions (GEMs) were dissolved and mixed. Subsequently, these products were purified, pre-amplified, and finally used for library construction.
Data quality control
Quality control of scRNA-seq data was conducted using the “Seurat” R package. Experimental and control samples were subjected to quality control and filtering, with specific criteria including a feature value greater than 300, a count value less than 100,000, and a mitochondrial gene content of less than 10%. Double cells were excluded based on a predicted rate of 4%. Subsequently, the data were normalized and then analyzed via principal component analysis (PCA) for the top 2,000 highly variable genes (HVGs). During the dimensionality reduction process, the batch effect was alleviated using the Harmony method.
Cell clustering and cell annotation
The average number of unique molecular identifiers (UMIs) across all cells was 3,658, and the mean number of genes per cell was 1,893. The average UMI percentage distribution in each cell was 0.0380. Significant principal components were selected for dimensionality-reduction analysis using Uniform Manifold Approximation and Projection (UMAP) to visualize gene expression. UMAP was employed to cluster cell subgroups. Preliminary annotation of these cell subgroups was performed using SingleR software, and manual annotation was carried out by integrating the results of automatic annotation. The cell types of each cluster were annotated, and cell subpopulations with significant differences between the experimental and control groups were screened.
Differential expression gene analysis and key gene screening
Differential gene screening
To identify the DEGs between IC and normal bladder tissues, differential expression analysis was conducted on the single-cell sequencing results of rat bladder tissue samples from the experimental and control groups. The analysis was performed using the DESeq2 package in R (version: 1.44.0).The threshold of differential expression analysis was set to |log2FC|>0.5, adjusted P < 0.05. Among them, those satisfying log2FC > 0.5 and adjusted P < 0.05 are up-regulated genes, and those satisfying log2FC<−0.5 and adjusted P < 0.05 are down-regulated genes.
Weighted gene co-expression network analysis
WGCNA is a powerful bioinformatics tool that can be utilized to identify groups of genes with a high degree of interconnectivity and to explore their associations with phenotypic features. In this study, WGCNA was applied to the DEG results. The power value of the proximity matrix weighting parameter was determined to be 10, and the scale independence was found to be 0.85. The samples in the dataset were clustered via WGCNA, with the Pearson correlation coefficient serving as the measure of similarity. After removing outliers, the samples were depicted in a clustering tree. The average degree of connectivity was set at 10. Subsequently, a scale-free topological network was constructed, enabling the identification of disease-related rhythmic gene clusters and immune gene clusters.
Core gene screening and PPI
Using the Venn2.1.0 online platform (https://bioinfogp.cnb.csic.es/tools/venny/), we determined the intersection of the rhythmic gene cluster and the immunity gene cluster to identify the hub genes involved in the occurrence and development of IC. The results were presented in a Venn diagram for clear visualization. The Protein-Protein Interaction (PPI) network was initially constructed with the STRING database. Subsequently, it was further refined using Cytoscape version 3.6.1. During the network construction process, only nodes with an interaction relationship confidence level exceeding 0.95 were incorporated, ensuring the reliability and significance of the network.
Functional enrichment analysis
Metascape was used to perform GO and KEGG functional enrichment analysis of differential genes to obtain relevant biological information on key targets. P < 0.01, minimum overlapping gene = 3, and minimum enrichment factor > 1.5 were taken as the criteria for the analysis. GO selected the top 10 information and KEGG selected the top 20 information. GO functional analysis classified the differentially expressed genes into cellular components, and analyzed functions and biological processes. KEGG pathway analysis analyzed the main pathways regulated by differentially expressed genes.
Functional verification experiments
RT-PCR
Total RNA was extracted from the rat bladders following the Trizol method, and its concentration was determined. Subsequently, reverse transcription was carried out using the Thermo reverse transcription kit to synthesize cDNA. The cDNA obtained from reverse transcription was diluted fivefold and then processed in accordance with the SYBGreen protocol. Real-time fluorescence quantitative PCR amplification was performed using the Thermo system with the following cycling conditions: an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 45 s. After amplification, the dissolution curve was generated. To normalize the expression of the rat target gene mRNA, GAPDH was used as an internal control, and the mean value of 2⁻ΔΔCt was calculated to represent the mRNA expression level of the TOP2A gene.
Western blot
Proteins were isolated by means of RIPA and PMSF. Subsequently, the protein concentration was quantified in accordance with the instructions provided by the BCA protein quantification kit. Afterward, the Western Blot protocol was followed. SDS-PAGE gels were fabricated using glass plates and gel holders. A total of 40µg of protein was loaded into each well, and a pre-stained protein Marker was added on both sides to serve as a molecular weight reference for electrophoresis, membrane transfer, and subsequent antibody incubation procedures. For primary antibody incubation, anti-GAPDH and anti-TOP2A antibodies were utilized respectively, and the samples were incubated overnight at 4 °C. Subsequently, secondary antibodies, namely the corresponding HRP-labeled secondary antibodies (goat anti-rabbit IgG or goat anti-mouse IgG), were incubated for 1 h at room temperature. Chemiluminescent detection was then carried out using an ECL luminescent solution, and images were acquired with a gel imaging system. Finally, the images were analyzed using ImageJ software, and the ratio of the gray value of the target protein band to that of the internal reference protein band was calculated to represent the relative expression level of the TOP2A protein.
Immunohistochemistry
Paraffin sections underwent a series of preparatory procedures. Firstly, they were dewaxed and rehydrated through the application of a dewaxing solution and graded ethanol. Subsequently, antigen repair was carried out by subjecting the sections to microwave treatment within a citrate buffer (pH 6.0). After natural cooling took place, the sections were thoroughly rinsed with PBS. Endogenous peroxidase activity was then blocked by the addition of a 3% hydrogen peroxide solution. Next, 3% BSA was utilized to block serum. Murine anti-CD4 + T cell antibody (at a dilution of 1:150, sourced from Abcam) and murine anti-CD8 + T cell antibody (at a dilution of 1:100, also from Abcam) were added to the blocked sections, which were then incubated overnight in a refrigerator set at 4 °C. The sections were rinsed with PBS buffer to remove the unbound primary antibodies in next day. Subsequently, a secondary antibody was added, and the sections were incubated at room temperature. The processed sections were then stained with a DAB working solution for a duration ranging from 15 to 30 s. Immediately after staining, the working solution was washed off to terminate the reaction. Hematoxylin nuclear staining was performed for 30 s, followed by a thorough wash with water to remove the staining solution. A gradient concentration of alcohol was employed for dehydration purposes, xylene was used to achieve transparency, and neutral gum was applied to seal the sections. Finally, the specimens were observed under a microscope.
CD4+T, CD8+T, TOP2A immunohistochemical detection score
The expression data of CD4 + T, CD8 + T, and TOP2A in both the experimental and control groups were collected. The scoring criteria for the percentage of positive cells were as follows: a percentage less than 5% was assigned a score of 0; between 6% − 25%, a score of 1; 26% − 50%, a score of 2; 51% − 75%, a score of 3; and 76% − 100%, a score of 4. Regarding colorability, no coloring was given 0 points, light tawny 1 point, dark tawny 2 points, and brown 3 points. Subsequently, the two scores were multiplied, and the results were categorized as follows: a product of 0–1 points was recorded as negative performance (-); 2–4 points as weakly positive performance (+); 5–8 points as positive performance (++); and 9–12 points as strongly positive performance (++++). The entire evaluation process was double-blind and interpreted by two associate senior or above-titled pathologists.
Statistical analysis
Statistical analysis of the scores related to CD4 + T, CD8 + T, and TOP2A was performed employing the R software and GraphPad Prism. A T-test was used for the comparison between the control group and the experimental group. Meanwhile, the Pearson correlation analysis was adopted to compute the correlation coefficients and explore the correlations. The significance level was preset at P < 0.05, meaning that only results with a probability less than 0.05 were considered statistically significant.
Results
The successful criteria for constructing the CYP-induced cystitis rat models
Von-Frey results
The results are shown in Fig. 2A-B.Compared with the control group, the mechanical pain threshold of the bladder of rats in the experimental group was significantly lower, and the response to Von-Frey force was significantly increased (P < 0.0001), the difference was statistically significant, indicating that the model preparation was successful.
Fig. 2. Results of animal model construction. A Bar graphs of pain response scores to different Von-Frey fiber filaments in two groups of rats; B Line graphs. *: P < 0.05; **:P < 0.01; ***: P < 0.001; ****: P < 0.0001; C urodynamic performance of rats in the control group; D urodynamic performance of rats in the experimental group; HE staining results of rats in the control group: E HE staining ⋅4; F HE staining ⋅10; G HE staining ⋅20; HE staining results of rats in the experimental group: H HE staining ⋅4; I HE staining ⋅10; J HE staining ⋅20. The HE staining results showed that the bladder tissues of the rats had bladder inflammation that It showed interstitial edema, marked inflammatory cell infiltration, lamina propria edema, submucosal hemorrhage, and focal damage to the uroepithelium
Results of urodynamic examination
As shown in Fig. 2C-D, after establishing the model, the time of the first bladder contraction and the first urination in the experimental group rats was significantly earlier than that in the control group, which was in line with the characteristics of early initial bladder contraction and early initial urination. In addition, compared with the control group, the micturition interval of the experimental group was significantly reduced (P < 0.0001), the maximal pressure of the urethra muscle was significantly increased (P < 0.0001), and the above differences were statistically significant, which indicated that the model preparation was successful, as shown in Table 1.
Table 1. Differences in urodynamic indexes between the two groups of ratsVariantControl group(n = 10)Experimental group(n = 10)t P MSDMSDMicturition interval(s)179.378.2277.476.6045.40<0.0001Maximum urethral pressure(cmH_2_0)27.674.8678.036.3830.51<0.0001
HE staining results
In Fig. 2E-G and 2H-J, the comparison shows that the bladders of rats in the experimental group exhibit obvious submucosal and lamina propria edema, hemorrhage, destruction of the bladder epithelium, thickening of the bladder epithelial layer. This finding strongly suggests that the model establishment was successful.
Single-cell sequencing results
Data quality control and cell annotation
Bladder single cells were isolated from the bladder tissues of both experimental and control rats and then underwent scRNA-seq. During this process, cells were screened based on specific criteria: a feature value exceeding 300, a count value less than 100,000, and a mitochondrial gene content lower than 10%. Additionally, double cells were removed in accordance with a predicted rate of 4%. Through strict quality control filtering to eliminate low-quality cells, a total of 8,658 cells were incorporated into the subsequent analysis. Specifically, there were 4,238 cells in the experimental group and 4,420 cells in the control group. The data that had passed the quality control filtering were clustered and analyzed. As a result, a total of 24 cell clusters were detected. Subsequently, these clusters were downscaled and projected using the UMAP plot method. Each cell subgroup was annotated as illustrated in Fig. 3A and could be classified into various cell clusters, namely T cells, B cells, epithelial cells, and endothelial cells.
Fig. 3. Single cell subpopulation and cellular annotation. A UMAP visualization results of each cell subpopulation in bladder single cells; B expression distribution of rhythmic topmarker genes in each cell subpopulation; C expression distribution of T-cell topmarker genes in each cell subpopulation; D annotated T-cell topmarker gene expression bubble map
Changes of T cells in control and CYP-induced cystitis rat bladders
Through a comparative analysis of the single-cell sequencing outcomes obtained from rats in the experimental group and those in the control group, it was discerned that substantial discrepancies existed in the cell classifications of the samples between the two groups. The single-cell sequencing data revealed that the relative abundance of T cells within the samples of the experimental group surpassed that of the control group. Consequently, T cells were designated for utilization in the subsequent experimental procedures of this study.
Results of WGCNA analysis of significant DEGs
A weighted gene co-expression network was constructed using average hierarchical clustering and dynamic tree clipping techniques. Through this process, a total of 18 modules were identified. Among these, the Immunity module and the Rhythm module demonstrated a higher correlation with interstitial cystitis. Consequently, these two modules were selected as key modules for subsequent analysis. Differential gene expression analyses were then separately conducted on the genes within these two modules. Genes with P < 0.05 and logFC ≥ 1 were screened as DEGs. As a result, 54 DEGs were obtained from the Immunity module, and 99 DEGs were identified from the Rhythm module.
Hub gene acquisition and PPI analysis
To screen the key genes, DEGs from the two significant modules were extracted, and their intersection was determined to generate a Venn diagram (Fig. 4A). Through this process, a single hub gene, TOP2A, was successfully screened. Additionally, six highly expressed genes were selected from among the marker genes in T cells: HMGB2, GAPDH, CORO1A, ACTG1, TUBA1B, and TOP2A. Notably, the expression level of TOP2A was significantly higher than that of the other five genes (Fig. 3B-D). The “GeneMania” tool was employed to predict the functions of key genes and potential interaction networks. Protein-protein interaction networks were separately constructed for the Immunity module and the Rhythm module. Intriguingly, both networks incorporated the hub gene TOP2A (Fig. 4B-C).
Fig. 4. Identification of TOP2A as a Hub Gene and Protein-Protein Interaction Networks in IC. A Results of taking the intersection of the Immunity and Rhythm modules; B hypothetical protein-protein interaction network composed of genes from the immunity module; C hypothetical protein-protein interaction network composed of genes from the rhythm module
GO and KEGG enrichment analysis
GO and KEGG pathway enrichment analyses were performed for the differentially expressed genes in the single-cell sequencing results to determine their possible biological functions, and the results of GO analysis showed that the biological functions of the differentially expressed genes were mainly enriched in aerobic respiration, cytoplasmic translation, oxidative phosphorylation, and proton motive force-driven mitochondrial ATP synthesis, and the major molecular functions of the differentially expressed genes were enriched in plasma membrane and ribosomes, and the major molecular functions were enriched in plasminogen. The main molecular functions were enriched in proton-translocated ATP synthase activity, oxidoreductase activity, calmodulin binding, etc.(Fig. 5A-B).KEGG functional analysis showed that the differentially expressed genes were mainly enriched in the ribosomes, oxidative phosphorylation, PPAR signaling pathway, biosynthesis of amino acids, glutathione metabolism, etc.(Fig. 5C-D). The results of enrichment analysis indicated that the differentially expressed genes might be involved in the occurrence and development of IC through the above biological mechanisms.
Fig. 5GO and KEGG enrichment analysis. A Bubble plots of GO enrichment analysis of differentially expressed genes; B GO gene-function association network diagram; C Bubble plots of KEGG enrichment analysis of differentially expressed genes; D KEGG gene-function association network diagram
Functional verification experiment results
TOP2A expression in CYP-induced cystitis rats
Western Blot and RT-PCR experiments were performed to detect the expression of TOP2A in rats from both the experimental and control groups. The results demonstrated that both the protein and mRNA expression levels of TOP2A in the experimental group rats were significantly higher than those in the control group (P < 0.001). (Fig. 6A-C)
Fig. 6. The protein expression level of TOP2A in the bladder of rats in the experimental group was significantly higher than that in the control group. A TOP2A and GAPDH protein expression in the bladder of control and experimental rats; B TOP2A protein expression in the control group was lower than that in the experimental rats; C TOP2A mRNA expression in the control group was lower than that in the experimental rats. : P < 0.001
Expression of TOP2A, CD4+T and CD8+T cells in CYP-induced cystitis rats
IHC results revealed that in the bladder tissues of rats in the experimental group, CD4+T cells exhibited a staining pattern of both the cell membrane and cytoplasm, distributed in the stroma; CD8 + T cells were also diffusely distributed in the stroma; while TOP2A was widely distributed in the stroma. *(Fig. 7A-I)*Through t-test for comparisons, it was demonstrated that, when compared to the control group, the expression levels of TOP2A, CD4 + T cells, and CD8 + T cells in the bladder tissues of rats in the experimental group were significantly elevated (P < 0.05), indicating a statistically significant difference. (Fig. 7J-K)
Fig. 7IHC results and expression levels of CD4 + T, CD8 + T, and TOP2A in two groups of rats. A, B, C Expression of CD4+ T cells in bladder tissues of CYP-induced cystitis rats; D, E, F Expression of CD8+ T cells in bladder tissues of CYP-induced cystitis rats; H, I, J Expression of TOP2A in bladder tissues of CYP-induced cystitis rats; J bar graph; K line graph. : P < 0.0001
Correlation analysis of TOP2A and CD4 + T and CD8 + T cell expression in CYP-induced cystitis rats
Pearson correlation analysis showed that TOP2A expression was significantly positively correlated with CD4 + T cell infiltration and moderately positively correlated with CD8 + T cell infiltration, suggesting that TOP2A may be involved in the immune microenvironmental regulation of IC through regulating the function of T cells (Fig. 8).
Fig. 8. Results of Pearson correlation analysis
Discussion
Interstitial cystitis (IC) is a complex chronic inflammatory disorder, and its pathogenesis is characterized by the dysregulation of multiple immune cells and molecular pathways [12, 13]. This study found through single-cell sequencing that in the CYP-induced cystitis model, T cells were significantly aggregated, suggesting that T cell-mediated immune responses may be a key component in the pathological development of IC. The findings revealed a significant increase in T cells, particularly CD4 + T and CD8 + T cells, in the experimental group, strongly indicating that T-cell-mediated immune responses play a pivotal role in the development of IC. Previous studies have demonstrated that in chronic inflammatory conditions, T cells contribute to tissue injury and fibrosis through the secretion of pro-inflammatory factors such as IFN-γ and TNF-α, as well as by mediating cytotoxic effects [14]. Although this study is based on an acute cystitis model, the TOP2A-mediated T cell infiltration phenotype shares similarities with the immune characteristics observed in human IC, warranting investigation into whether this mechanism may play an important role in bladder inflammation.
From a mechanistic perspective, the upregulation of TOP2A expression is significantly positively correlated with the degree of T cell infiltration, suggesting that it may act as an upstream molecule regulating T cell function. We hypothesize that TOP2A promotes the amplification and persistence of inflammatory signals by affecting T cell proliferation and functional differentiation, thereby exacerbating immunopathological damage in bladder tissue. On the other hand, the inflammatory microenvironment shaped by activated T cells may feedback and regulate TOP2A expression in local stromal cells through paracrine mechanisms, forming a positive regulatory loop that jointly drives disease progression. Immunohistochemical results showed that CD4 T cells exhibited a membranous co-staining pattern in the bladder stroma, and their spatial distribution was consistent with cytokine secretion function; CD8 T cells were diffusely distributed in the stroma. These findings recapitulate aspects of previous studies on the distribution and function of T cell subsets in the bladder tissues of IC patients [15–17]. Further correlation analysis showed that TOP2A expression was positively correlated with CD4 T cell infiltration (r = 0.89) and CD8 T cell infiltration (r = 0.64), statistically supporting a close association between TOP2A and T cell function. These results imply that TOP2A may participate in the regulation of the IC immune microenvironment by modulating T cell function. Notably, TOP2A, a DNA topoisomerase, is known to be involved not only in cell cycle regulation but also in immune cell activation and differentiation [18]. For instance, in rheumatoid arthritis, elevated TOP2A expression is linked to abnormal T cell proliferation and increased production of inflammatory factors [19]. Based on these findings, we speculate that during the pathogenesis of IC, TOP2A may drive the local immune microenvironment toward a pro-inflammatory direction by regulating T cell proliferation dynamics and functional polarization states, while the inflammatory microenvironment may, in turn, further regulate the expression levels of TOP2A by altering the epigenetic status or transcriptional activity of local cells, forming a positive feedback loop that sustains chronic inflammation.
In recent years, the role of rhythmic genes in regulating immune responses and inflammatory processes has attracted growing attention. Biological rhythms, particularly the rhythmic genes, modulate immune cell function and the secretion of inflammatory factors by controlling the cyclic expression of genes [20, 21]. The GO enrichment analysis conducted in this study indicates that TOP2A may influence the regulation of neuronal death, as well as cellular processes such as respiration, migration, and epithelial cell proliferation. Some researchers have discovered that the activation of the immune system is regulated according to the time of day through the deciphering of immune-response-related cell migration mechanisms [22]. This finding implies that the TOP2A gene may contribute to immune system abnormalities, ultimately leading to inflammation. Furthermore, the function of TOP2A in sensory neuromodulation and neurotransmitter release, along with its interactions with mast cells and inflammatory mediators, may be associated with the chronic and neurogenic inflammation underlying IC symptoms, including bladder pain and urgency. These symptoms are linked to dysregulated pathways influenced by factors such as immune dysfunction and infection. Our observation of a significant upregulation of the rhythmic gene TOP2A in the CYP-induced cystitis model implicates its potential role in the disease process, meriting further investigation in human IC. Related research has demonstrated that fluctuations in the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, during the late night and early morning hours are closely associated with circadian rhythms [23]. Moreover, the circadian oscillations of cytokines may be regulated by rhythmic genes [24], while pro-inflammatory cytokines can also contribute to rhythm disturbances [25, 26]. The vicious cycle formed by the interaction between rhythm disturbances and pro-inflammatory cytokines is likely one of the key pathogenic factors in chronic inflammatory diseases. Additionally, the chronic bladder pain experienced by IC patients can cause depression and anxiety by disrupting sleep [27], and this symptom may be related to rhythm disturbances. Previous studies have shown that TOP2A expression is regulated by biological rhythms and exhibits cyclic changes in inflammatory diseases [28]. Integrating these findings with the results of the present study, we propose a hypothesis: TOP2A may be involved in the pathogenesis of IC by regulating the rhythmic functions of T cells, including their proliferation, migration, and secretion of inflammatory factors. Additionally, TOP2A may further exacerbate immune microenvironmental disruptions in IC by affecting the functions of other immune cells, such as macrophages and dendritic cells. However, the precise regulatory mechanism of TOP2A in IC remains to be elucidated. Future studies could involve knockdown or overexpression experiments to validate its role in T cell function regulation and explore whether it influences immune responses through interactions with other rhythmic genes.
The current study is not without limitations. A primary limitation of this study is the use of an acute CYP-induced cystitis model, which may not fully recapitulate the chronicity and complexity of human IC. Therefore, our findings regarding TOP2A in the CYP-induced cystitis model reveal a potential mechanism, motivating future studies to confirm its upregulation and function in human IC tissues. If validated, TOP2A could emerge as a promising therapeutic target for modulating the aberrant immune response in IC. Moreover, additional experimental evidence and targeted clinical trials are required to elucidate the long-term impact of circadian rhythm disruption on IC, which would provide a more comprehensive understanding of the disease mechanism and guide therapeutic development.
In summary, our single-cell sequencing analysis of CYP-induced cystitis reveals notable T-cell infiltration and its association with TOP2A, thereby laying the groundwork for future studies to explore this rhythmic gene as a potential immunomodulator in human IC. Specifically, TOP2A is likely to participate in the development of CYP-induced cystitis by modulating T cell function, providing insights that may be relevant to IC. For future research directions, it is imperative to delve deeper into the specific regulatory mechanisms of TOP2A in IC and explore its clinical application potential. For instance, conducting pre-clinical trials that involve pharmacological intervention to modulate the expression or function of TOP2A, and meticulously observing its impact on the pathological progression of IC. Additionally, integrating multi-omics analyses, including epigenetics and metabolomics, may facilitate a more comprehensive understanding of the functional network of TOP2A in IC. Such an approach could potentially offer innovative insights for the precision treatment of this complex disease, enhancing the effectiveness and specificity of therapeutic interventions.