Molecular Profiling of Inflammatory Processes in a Mouse Model of IC/BPS: From the Complete Transcriptome to Major Sex-Related Histological Features of the Urinary Bladder

Abstract

Animal models are invaluable in the research of the pathophysiology of interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic aseptic urinary bladder disease of unknown etiology that primarily affects women. Here, a mouse model of IC/BPS was induced with multiple low-dose cyclophosphamide (CYP) applications and thoroughly characterized by RNA sequencing, qPCR, Western blot, and immunolabeling to elucidate key inflammatory processes and sex-dependent differences in the bladder inflammatory response. CYP treatment resulted in the upregulation of inflammatory transcripts such as Ccl8, Eda2r, and Vegfd, which are predominantly involved in innate immunity pathways, recapitulating the crucial findings in the bladder transcriptome of IC/BPS patients. The JAK/STAT signaling pathway was analyzed in detail, and the JAK3/STAT3 interaction was found to be most activated in cells of the bladder urothelium and lamina propria. Sex-based data analysis revealed that cell proliferation was more pronounced in male bladders, while innate immunity and tissue remodeling processes were the most distinctive responses of female bladders to CYP treatment. These processes were also reflected in prominent histological changes in the bladder. The study provides an invaluable reference dataset for preclinical research on IC/BPS and an insight into the sex-specific mechanisms involved in the development of IC/BPS pathology, which may explain the more frequent occurrence of this disease in women.

Keywords: RNA sequencing; animal model; cyclophosphamide; inflammation; interstitial cystitis/bladder pain syndrome.

Figure 1

Representation of study workflow. Created with www.biorender.com (accessed on 20 January 2023).

Figure 2

Effects of CYP treatment on the complete transcriptome of mouse bladders. (A) A volcano plot showing upregulated (red), downregulated (green), and unchanged (blue) genes in CYP vs. Ctrl mice. (B) Heatmap showing the 1000 most significantly DEGs in CYP (yellow; n = 6) vs. Ctrl (green; n = 6) animals (p adj < 0.05). Both rows and columns are clustered using correlation distance and average linkage. Lower expression is indicated in blue, and higher expression is indicated in red. CM: Ctrl male; CF: Ctrl female; TM: CYP-treated male; TF: CYP-treated female. (C) PCA of the 1000 most significantly DEGs in CYP (blue; n = 6) vs. Ctrl (red; n = 6) animals (p adj < 0.05), showing principal components 1 and 2 (PC1 and PC2) explaining 70.7% and 7.5% of the total variance, respectively. Unit variance scaling was applied to rows, and singular value decomposition (SVD) with imputation was used to calculate principal components. The prediction ellipses are such that with a probability of 0.95, a new observation from the same group will fall inside the ellipse. (D) KEGG enrichment analysis based on all DEGs in CYP (n = 6) vs. Ctrl (n = 6) animals (p adj < 0.05), showing relevant significantly enriched pathways, represented as −log10 of the p adj value. Processes involved in cell proliferation are circled in gray, while processes of immune response are circled in pink.

Figure 3

Effects of CYP treatment on the innate immune response in mouse bladders. (A) Heatmap showing DEGs (n = 89) with p adj < 0.05 detected in the enriched KEGG pathways of innate immunity response comparing the CYP (yellow; n = 6 animals) vs. Ctrl (green; n = 6 animals) groups. Both the rows and columns are clustered using correlation distance and average linkage. Lower expression is marked in blue; higher expression is marked in red. CM: Ctrl male; CF: Ctrl female; TM: CYP-treated male; TF: CYP-treated female. (B) A graphical representation of the five most significantly DEGs in each enriched pathway of innate immunity. Shown are the −log10 of the p adj values of the transcripts in the CYP vs. Ctrl group of animals. Circled are DEGs enriched in more than one pathway. (C) qPCR validation of the selected DEGs shown in (B), confirming their upregulation in the CYP vs. Ctrl group. Shown are the means ± SD of negative Δ Ct determined for 10 animals per group. The qPCR analysis was not reliable for some DEGs (Tnfrsf10b, Gdf15, Ulbp1, Casp3, Raet1d, Casp7, Masp1, Vsig4, Bdkrb1, C4b, Il6, Spp1, Cdkn1a, Il7), therefore the results are not presented. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

The JAK/STAT signaling pathway is activated in the bladders of CYP-treated animals. (A) Heatmap showing DEGs (n = 37) with p adj < 0.05 detected in the JAK/STAT signaling pathway by KEGG analysis comparing the CYP (yellow; n = 6 animals) vs. Ctrl (green; n = 6 animals) groups. Both the rows and columns are clustered using correlation distance and average linkage. Lower expression is marked in blue, and higher expression is marked in red. CM: Ctrl male; CF: Ctrl female; TM: CYP-treated male; TF: CYP-treated female. (B) qPCR validation of mRNA expression for cytokines and cytokine receptors (Il13ra2, Il13ra1, Il11), comparing CYP and Ctrl animals. Shown are the mean ± SD of negative Δ Ct determined for 10 animals per group. The qPCR analysis was not reliable for some DEGs identified as cytokines and cytokine receptors in the JAK/STAT signaling pathway (Il6, Il6ra, Il7, Il21r, Il3ra), therefore the results are not presented. (C) qPCR validation of mRNA expression for inhibitors of JAK/STAT signaling (Socs2, Socs3, Ptpn6), comparing CYP and Ctrl animals. Shown are the means ± SD of negative Δ Ct determined for 10 animals per group. (D) qPCR validation of transcript expression for the analyzed JAK and STAT molecules (Jak1, Jak3, Stat3), comparing CYP and Ctrl animals. Shown are the means ± SD of negative Δ Ct determined for 10 animals per group. (E) Expression of JAK and STAT proteins (JAK1, JAK3, and STAT3), determined by Western blot in Ctrl and CYP animals. Shown are the mean ± SD of relative protein expression normalized to β-actin for six animals per group. (F) The expression of phosphorylated JAK and STAT proteins (pJAK3, pSTAT3), determined by Western blot in Ctrl and CYP animals. Shown are the mean ± SD of relative protein expression normalized to β-actin for six animals per group. (G) Representative blots for the analysis of protein expression in six animals per group. * p < 0.05; *** p < 0.001.

Figure 5

Representative confocal microscopy images of pSTAT3 nuclear translocation in the bladders of CYP-treated mice, selected from a series of images assembling a Z-stack. (A) Nuclear expression of pSTAT3 in superficial urothelial cells (white arrows) and interstitial cells of ULP (yellow arrows). A cut-view of the Z-stack shows pSTAT3 immunoreactions in the nucleus of a superficial urothelial cell (white asterisk) and an interstitial cell of the ULP (yellow asterisk). The dotted line indicates the urothelial basal lamina. (B) Nuclear expression of pSTAT3 in endothelial cells (white arrows) and the interstitial cells of DLP (yellow arrows). The Z-stack cut-view shows pSTAT3 immunoreactions in the nucleus of the endothelial cell (white asterisk) and interstitial cell of DLP (yellow asterisk). The dotted line indicates the endothelial basal lamina. (C) Colocalization of pSTAT3 and F4/80 immunoreactions in some of the interstitial cells of DLP (white arrows). A cut-view of the Z-stack shows pSTAT3 immunoreaction in the nuclei of macrophages (white asterisk). Yellow arrows point to interstitial cells that were not identified as macrophages. The dotted line indicates the endothelial basal lamina. (D) Colocalization of pSTAT3 and vimentin immunoreactions in some of the interstitial cells of DLP (white arrows). A cut-view of the Z-stack shows pSTAT3 immunoreactions in vimentin-positive mesenchymal cells (white asterisks). The yellow arrow shows an interstitial cell of non-mesenchymal origin. The dotted line indicates the endothelial basal lamina. Scale bars: 20 µm.

Figure 6

The CYP-induced enrichment of different processes is sex-specific. (A) GO enrichment analysis showing significantly enriched biological processes (blue), cell components (yellow), and molecular functions (green). Represented are −log10 p adj values for each process, comparing CYP males (dashed bars) and CYP females (empty bars). Numbers at the end of each bar represent the fold change between CYP males and CYP females in the p adj value of each enriched process. (B) KEGG enrichment analysis showing significantly enriched pathways (−log10 of p adj) comparing CYP males (green bars) and CYP females (orange bars). Numbers at the end of each bar represent the fold change between CYP males and CYP females in the p adj value of each enriched process. Processes involved in cell proliferation are circled in gray; processes of cell-extracellular space communication are circled in pink; and processes of innate immunity are circled in yellow.

Figure 7

CYP-induced changes in the bladders of female mice. (A) STRING network shows interactions between the putative proteins of the significantly DEGs identified in the enriched GO cellular components of the extracellular region, extracellular matrix, and extracellular region part (n = 56), displaying only interactions of high confidence (0.7). The proteins are known and predicted to interact in immune system processes (green), tissue remodeling (red), and EMC organization (blue). Disconnected nodes are hidden. (B) The mRNA expression of Ccl8 shows significant upregulation of the transcript in females after CYP treatment. Shown are the means ± SD of negative Δ Ct determined for five animals per group. (C) Differences in expression of Ccl8 mRNA in males and females after CYP treatment. The fold change of average Δ Ct in Ctrl vs. CYP males and Ctrl vs. CYP females is represented on the y axis. The number written above the bars indicates the fold change in mRNA expression in females vs. males after CYP treatment. (D) mRNA expression of Ccr1 and Ccr5 demonstrates a greater increase in females compared to males after CYP treatment. Shown are the mean ± SD of normalized read counts determined by RNA seq for three animals per group. (E) mRNA expression of selected proteinases (Mmp2, Mmp14, Mmp19, Mmp23, Mmp28, Adamts1, Adamts5) demonstrates a greater increase in females before and after CYP treatment compared to males. Shown are the mean ± SD of normalized read counts, determined by RNA seq for three animals per group. Blue lines indicate the fold change in average read counts between the groups (Ctrl males vs. Ctrl females and CYP males vs. CYP females). (F) Representative images of HE-stained bladder sections showing the difference in LP thickness between Ctrl female, CYP female, and CYP male. Dotted-line borders the measured surface area of LP. Scale bars: 250 µm. (G) Quantitative analysis of LP thickness reveals a greater thickness of LP in females compared to males before and after CYP treatment. Blue lines indicate the fold change in average LP thickness between the groups (Ctrl males vs. Ctrl females and CYP males vs. CYP females). * p < 0.05; ** p < 0.01.

Figure 8

CYP-induced changes in the bladders of male mice. (A) STRING network shows interactions between the putative proteins of the significantly DEGs identified in the enriched GO biological processes involved in cell proliferation (n = 76), displaying only interactions of the highest confidence (0.9). The proteins are known and predicted to interact during DNA replication (red) and cell division (blue). Disconnected nodes are hidden. (B) Left part of the panel: mRNA expression of Mki67 showing more significant upregulation of the transcripts in males compared to females after CYP treatment. Shown are the means ± SD of negative Δ Ct determined for five animals per group. Right part of the panel: fold change of Mki67 mRNA expression in males and females after CYP treatment. (C) Representative images of Ki67 immunolabeled sections of the bladder wall of Ctrl male, CYP female, and CYP male show the differences in the distribution of Ki67-positive nuclei in the bladder wall. The dotted line represents the basal lamina. L; lumen of the bladder. Scale bar: 100 µm. * p < 0.05; ** p < 0.01.