Radiation of the urinary bladder attenuates the development of lipopolysaccharide-induced cystitis

https://doi.org/10.1016/j.intimp.2020.106334Get rights and content

Highlights

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    Bladder irradiation inhibited LPS-induced changes in mast cells and Iba1 + cells.

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    Bladder irradiation potentiated LPS-driven oxidative stress.

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    Radiation may change the bladder immune response to danger signals.

Abstract

In the present study we assessed how ionizing radiation affects TLR4-stimulated immune activation in lipopolysaccharide (LPS)-induced cystitis. LPS or saline was administered intravesically to female rats followed by urinary bladder irradiation (20 Gy) 24 h later or sham treatment. Presence in the urinary bladder of inflammatory cells (mast cells, CD3+, ionized calcium-binding adapter molecule 1 (Iba-1)+, CD68+, CD40+, CD80+, CD11c + and CD206 + cells) and expression of oxidative stress (8-OHdG), hypoxia (HIF1α) and anti-oxidative responses (NRF2, HO-1, SOD1, SOD2, catalase) were assessed 14 days later with western blot, qPCR and/or immunohistochemistry. LPS stimulation resulted in a decrease of Iba-1 + cells in the urothelium, an increase in mast cells in the submucosa and a decrease in the bladder protein expression of HO-1, while no changes in the bladder expression of 8-OHdG, NRF2, SOD1, SOD2, catalase and HIF1α were observed. Bladder irradiation inhibited the LPS-driven increase in mast cells and the decrease in Iba1 + cells. Combining LPS and radiation increased the expression of 8-OHdG and number of CD3-positive cells in the urothelium and led to a decrease in NRF2α gene expression in the urinary bladder. In conclusion, irradiation may attenuate LPS-induced immune responses in the urinary bladder but potentiates LPS-induced oxidative stress, which as a consequence may have an impact on the urinary bladder immune sensing of pathogens and danger signals.

Introduction

Radiotherapy against tumours of the pelvic viscera such as of the prostate, rectum and uterine cervix constitutes an important part in the oncological treatment arsenal. This therapeutic approach is, however, associated with side effects such as cystitis and proctitis, since radiotherapy affects not only the pelvic tumour but also normal tissue in its vicinity. Acute side effects due to bladder irradiation are common and include frequency and urgency, dysuria and bleeding [1]. However, the current understanding is limited regarding what happens in normal tissue in the pelvic area in response to irradiation.

Toll-like receptors (TLRs) constitute important factors within the innate immune response and respond to different pathogens and tissue damage by activating the immune system. Previous studies in transgenic mice have shown that TLR4 contributes to radioresistance [2]. TLR4 in the urothelium is activated by lipopolysaccharide (LPS) and activates an intracellular response involving NF-κB, triggering the release of interleukin 6 (IL-6) and interleukin 8 (IL-8) [3], [4], [5]. We have previously shown that two weeks after bladder irradiation the expression of TLR4 is down-regulated in the urothelium and nitrergic pathways and IL-6 production are suppressed and IL-10 is up-regulated in the urinary bladder [6]. The effects of irradiation were more consistent with an anti-inflammatory response than a pro-inflammatory response two weeks after bladder irradiation.

HIF1α is induced by hypoxia, which leads to the transcription of genes controlling oxidative stress and genes encoding proteins stimulating angiogenesis such as vascular endothelial growth factor (VEGF) [7]. In radiation-induced cystitis, a progressive obliteration of small vessels occurs, which leads to tissue damage due to hypoxia [8]. HIF1α and downstream VEGF may be up-regulated in response to tissue hypoxia induced by radiation [9]. However, reactive oxygen species (ROS) may also stabilize the expression of HIF1α during hypoxia [10]. Moreover, HIF1α may be up-regulated by ROS in the tumour reoxygenation phase following radiotherapy [11]. NRF2 is a transcription factor regulating the transcription of many genes including heme oxygenase 1 (HO-1) involved in the antioxidative response [12], [13]. HO-1 may also be upregulated under hypoxic conditions by HIF1α [14]. In our previous report we showed that oxidative stress and the anti-oxidative regulators NF-E2-related factor 2 alpha (NRF2α), HO-1 and superoxide dismutase 2 (SOD2) were increased in the urinary bladder 28 days after bladder irradiation [15].

We hypothesized that bladder irradiation may lead to a changed immunological response to inflammatory inputs. In an animal model of LPS-induced cystitis we therefore tested how bladder irradiation affects LPS-induced changes in inflammation and oxidative stress.

Section snippets

Urinary bladder irradiation

The study was approved by the Animal Ethics Committee of University of Gothenburg. Female rats of the Sprague-Dawley strain with weights of 200–250 g were anaesthetized with ketamine (50 mg/kg intramuscularly) and medetomidine (10 µg/kg intraperitoneally). A thin catheter was then inserted per urethram into the bladder, urine was voided and LPS (100 µg/ml) or saline (control) was instilled for 60 min. Twenty-four hours later, rats were sedated and placed in the supine position and legs were

Histology and presence of inflammatory cells

LPS pre-treatment decreased the absolute number of urothelial cells in control rats (p < 0.05; n = 6–8; Fig. S1b). In irradiated rats, LPS pre-treatment increased the urinary bladder weight (337 ± 22 mg/kg vs. 252 ± 13 mg/kg for LPS-rad rats and Sal-rad rats, respectively, p < 0.05, n = 5–15; Fig. S1d) and increased the number of proliferating cells in the submucosa (p < 0.05; n = 6–8; Fig. S1g). CD3 + lymphocytes were present in the submucosa and urothelium in Sal-ctrl rats. While radiation

Discussion

This study demonstrates that radiation may affect the development of LPS-induced cystitis. On one hand, radiation blocked LPS-induced changes in inflammatory cells in the urinary bladder. On the other hand, radiation augmented oxidative stress in the urinary bladder of rats pre-treated with LPS.

As in our previous study [19], it is shown that LPS may induce inflammatory changes in the urinary bladder without the need to rupture mechanically or enzymatically the integrity of the urothelium [20],

Author statement

LP, LN and DG designed the study, LP, MFM, AJD, ÅTN and DG performed the experiments, LP, MFM, AJD, ÅTN and DG performed the analyses and LP and DG wrote the manuscript with input from all authors.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

The authors are thankful to the staff at the radiation facility, Department of Oncology, the Sahlgrenska University Hospital, for assistance during irradiation of the rats and to laboratory technician Anna-Lena Leverin, Department of Physiology, Sahlgrenska Academy at the University of Gothenburg for help with western blot analyses. The authors are also thankful to Histo-Center, Västra Frölunda, Sweden, for help with bladder tissue preparations and to Core Facilities, University of Gothenburg,

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