Interleukin-1 receptor antagonist overexpression in mesenchymal stem cells improves hemorrhagic cystitis outcomes via HtrA serine peptidase 3

Abstract

Background: Hemorrhagic cystitis (HC), a frequent complication of hematopoietic stem cell transplantation (HSCT), significantly affects quality of life and may worsen prognosis. Mesenchymal stem cells (MSCs) are known for their anti-inflammatory and tissue-regenerative properties. IL-1 receptor antagonist (IL-1Ra) blocks IL-1α and IL-1β by binding IL-1 receptors, offering potential therapeutic benefits. The aim of this study was to explore the therapeutic effect of MSCs overexpressing IL-1Ra on HC and investigate the underlying mechanisms.

Methods: MSCs were isolated from human umbilical cord tissues, and IL-1Ra-overexpressing MSCs (oeIL-1Ra-MSCs) were generated using lentiviral transfections. HC was induced in rats by cyclophosphamide administration. Rats received tail vein injections of either oeIL-1Ra-MSCs or control MSCs (Mock-MSCs). Hematuria and bladder tissue changes were assessed using test strips and hematoxylin & eosin (HE) staining. Immunohistochemistry detected molecular changes in bladder tissues. Gene expression differences between the two MSC groups were analyzed by mRNA sequencing and ChIP techniques.

Results: Treatment with oeIL-1Ra-MSCs significantly alleviated hematuria and reduced bladder edema and hemorrhage, and reduced mRNA expression levels of IL-1β, IL-6, and TNF-α in bladder tissues, compared with those in the Mock-MSC treatment group. Immunohistochemical staining showed a higher presence of CD105-positive cells (a marker for human MSCs) and CD31-positive vessels in bladder tissues treated with oeIL-1Ra-MSCs, indicating enhanced MSC migration and vascular stability. In vitro migration assay demonstrated a higher migration capacity of IL-1Ra overexpressing MSCs compared with that of control MSCs. Moreover, angiopoietin-1 (Ang-1) expression increased, while Angiopoietin-2 (Ang-2) expression decreased in bladder tissues treated with oeIL-1Ra-MSCs, suggesting enhanced blood vessel stabilization. Conditioned medium from oeIL-1Ra-MSC cultures stimulated human umbilical vein endothelial cell (hUVEC) migration, proliferation, and angiogenesis more effectively compared with that in control MSCs. mRNA sequencing revealed elevated HtrA3 expression in oeIL-1Ra-MSCs compared with that in control MSCs. Molecular analysis suggested that IL-1Ra overexpression in MSCs upregulated HtrA3 expression through inhibition of the JNK-c-Jun pathway and activation of the ERK-Egr-1 pathway.

Conclusion: Overexpression of IL-1Ra significantly enhances the therapeutic efficacy of MSCs in HC by promoting MSC migration to damaged bladder tissues, suppressing inflammation, stabilizing blood vessels, and upregulating angiogenesis via activation of HtrA3 signaling pathways.

Keywords: Hemorrhagic cystitis; HtrA serine peptidase 3; Interleukin-1 receptor antagonist; Mesenchymal stem cell; Migration.

Fig. 1

oeIL-1Ra-MSCs significantly alleviated hematuria and bladder oedema in HC rats. A Representative hematuria test-strip presentations for each group in the same experiment. B Hematuria test-strip score collected from two experiments and performed statistics (n = 3). C Naked eye view of a rat bladder. D Wet weight of rat bladders (n = 6). E HE staining of rat bladder tissues and rat bladder tissue hemorrhagic edema scores based on HE staining (n = 3). F Stained rat bladder tissue for CD105 expression and the number of CD105-positive cells in bladder tissue of rats treated with Mock-MSCs or oeIL-1Ra-MSCs on Day 7 (n = 3). G Transwell assays conducted to detect differences in the migratory abilities of Mock-MSCs and oeIL-1Ra-MSCs. Scale bar = 100 μm or 50 μm. Data are presented as the mean ± standard error of the mean (SEM) of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

oeIL-1Ra-MSCs attenuated the inflammatory response in bladder tissues from HC rats. A CD80 staining of rat bladder tissues and CD80-positive cell counts (n = 3). B CD206 staining of rat bladder tissues and CD206-positive cell counts (n = 3). C–E mRNA expression levels of IL-1β, IL-6, and TNF-α in rat bladder tissues (n = 3). Scale bar = 50 μm. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

oeIL-1Ra-MSCs promoted angiogenesis and stabilized blood vessels. A CD31 staining of labeled blood vessels and the number of blood vessels in rat bladder tissues (n = 3). B Ang-1 staining in rat bladder tissue and its positive areas (n = 3). C Ang-2 staining in rat bladder tissue and Ang-2-positive areas (n = 3). D and E Numbers of migrating hUVECs following culture in control medium, Mock-MSC-conditioned medium, or oeIL-1Ra-MSC-conditioned medium analyzed in Transwell cell-migration assays (n = 3). F mRNA-expression levels of Zo-1 in rat bladder tissues (n = 3). G–I Angiogenesis of hUVECs after culture in blank medium, Mock-MSC-conditioned medium, or oeIL-1Ra-MSC-conditioned medium, and the number of vessels generated at 2h, 3h (n = 3). J–L Edu proliferation assays performed to analyze the proportion of Edu-positive cells in hUVECs cultured with blank medium, Mock-MSC-conditioned medium, or oeIL-1Ra-MSC-conditioned medium at 12h, 24h (n = 3). Scale bar = 50 μm. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Overexpressing IL-1Ra in MSCs increased HtrA3 expression. A Volcano plot showing differentially expressed genes in Mock-MSCs and oeIL-1Ra-MSCs. B Heat map showing upregulated and downregulated genes in Mock-MSCs and oeIL-1Ra-MSCs. C HtrA3 mRNA expression in Mock-MSCs and oeIL-1Ra-MSCs (n = 3). D and E HtrA3 protein expression in Mock-MSCs and oeIL-1Ra-MSCs (n = 3). F HtrA3 secretion from Mock-MSCs and oeIL-1Ra-MSCs into their supernatants (n = 3). G and H HtrA3 staining of rat bladder tissues and statistical analysis of the positive areas (n = 3). I HtrA3 mRNA expression in rat bladder tissues after treatment with Mock-MSCs or oeIL-1Ra-MSCs (n = 3). Scale bar = 50 μm. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001

Fig. 5

HtrA3 knockdown reversed the effects of oeIL-1Ra-MSCs in vitro and in vivo. A–C HtrA3 expression at the mRNA and protein levels in Mock-MSCs, oeIL-1Ra-MSCs, and oeIL-1Ra-MSCs transfected with siHtrA3. D Numbers of migrating Mock-MSCs, oeIL-1Ra-MSCs, and oeIL-1Ra-MSCs transfected with siHtrA3 measured in Transwell cell-migration assays (n = 3). E–G Expression of HtrA3 at the mRNA and protein levels in hUVECs after culture with blank medium, Mock-MSC-conditioned medium, oeIL-1Ra-MSC-conditioned medium, or oeIL-1Ra-MSCs+siHtrA3-conditioned media (n = 3). H HUVECs were incubated with blank medium, Mock-MSCs-conditioned medium, oeIL-1Ra-MSCs-conditioned medium, or oeIL-1Ra-MSCS+siHtrA3-conditioned medium for 24 h. Angiogenesis was observed after 2 and 3 h, and the number of vessels was counted. Scale bar = 50 μm (n = 3). (I and J) Hematuria test strips and rat bladders after treatment with Mock-MSCs, oeIL-1Ra-MSCs, or oeIL-1Ra-MSCs transfected with siHtrA3. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001

Fig. 6

oeIL-1Ra-MSCs promoted high HtrA3 expression by inhibiting the c-Jun pathway and activating the ERK–Egr-1 pathway. A Nuclear p-c-Jun protein expression relative to endogenous reference histone-3 protein expression. B Total protein p-JNK and HtrA3 levels relative to endogenous GAPDH expression. C Total cellular p-ERK, Egr-1, and HtrA3 protein levels measured relative to endogenous GAPDH levels, with or without ravoxertinib treatment. D Predicted sites in the promoter region of the HtrA3 gene binding to Egr-1. E Dual luciferase reporter-gene assays detected Egr-1-dependent initiation of HtrA3 transcription. F ChIP followed by PCR was performed to detect Egr-1 binding to HtrA3. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 7

Mechanisms whereby IL-1Ra overexpression in MSCs significantly improved the prognosis of HC through the HtrA3 signaling pathway. oeIL-1Ra overexpression in MSCs increased the ability of MSCs to migrate to damaged tissues, and secreted IL-1Ra inhibited inflammation in HC. In addition, oeIL-1Ra-MSCs inhibited the JNK–c-JUN pathway to promote HtrA3 expression and activated the ERK–Egr-1 pathway to initiate the transcription and high expression of HtrA3, which promoted angiogenesis and MSC migration