Interstitial cystitis (IC), referred to bladder pain syndrome, characterized by intense pelvic pain and urinary symptoms, is a severely debilitating and chronic disorder. The etiology and pathophysiology of IC still remain an enigma, which makes the diagnosis difficult and treatment challenging. The current therapies of IC showed limited effects and relatively high recurrence rates in the long-term follow-up. About 10% of the diagnosed patients have to receive destructive surgeries (augmentation ileocystoplasty, urinary diversion, etc.) followed by stepwise therapeutic approaches, and 20% of whom have to face the failures (reference 1). Therefore, there is a pressing need to understand the molecular mechanisms underlying the IC development and to identify more efficient targets for therapeutic treatments. 
There have been several theories suggesting the causes of IC, including inflammation, neural changes, defects in the wall of bladder and activated mast cells, but none of which has fully explained the manifestations of this disease. Although no one could determine whether IC is an autoimmune disease, the current evidence shows that immunity might play an important role in the progress of IC (reference 2). Thus, we aimed to investigate the immune landscape (the distribution of immune cell subsets) in IC bladders and the specific stage in which the immunity might involve in the progress of IC. Single-cell RNA sequencing (scRNA-Seq), with the ability to reveal distinct subpopulations among cell types, has emerged as a powerful tool to capture the complex profiles of immune infiltrations in diseases. Integrating scRNA-seq with spatial transcriptomic (ST) has been applied to uncover the tissue architecture in pancreatic ductal adenocarcinomas (reference 3). To better understand the role of immunity in IC bladders, we firstly conducted a comprehensive phenotypic and functional investigation of immune parameters (using only CD45+ single cells) through scRNA-Seq combining mass cytometry (CyTOF). Then, the immune views resulted from scRNA-Seq were integrated with the results of ST through multimodal intersection analysis. The findings of this study reveal the immune landscape of bladder in IC patients and may pave the way for future studies of pathophysiology and therapy.

This prospective study was designed to investigate the immune atlas of human bladder of female patients with IC. Human research was approved by our Medical Ethics Committee. The study was performed after obtaining informed consent from all participants. The diagnosis of IC was in line with the National Institute of Diabetes, Digestive and Kidney Diseases guidelines. The characteristic pathological findings in the bladder wall were identified by cystoscopy. The inclusion criteria and exclusion criteria for IC patients were shown as follow. 

Inclusion criteria	
  1) Patients of 18 years of age or older at the time of informed consent;
  2) Previously diagnosed with interstitial cystitis/bladder pain syndrome (IC/BPS) for a duration of >6 months;
  3) Currently diagnosed with Hunner type interstitial cystitis by cystoscopy;
  4) O’Leary-Sant Interstitial Cystitis Symptom and Problem Index score over 18;
  5) Understands the purpose of this study as explained by the investigator, and that their participation is voluntary and they are free to withhold consent or withdraw from the study at any time, and the investigators determined that the patient is suitable for participation in the study.
	
Exclusion criteria	
1. General conditions	
  1) The investigators determined she was not suitable for the study;
  2) Currently diagnosed with cancer, or have previous history of cancer within the preceding 5 years;
  3) Currently diagnosed with severe heart, lung, liver, kidney, or blood disorder;
  4) Patients who are pregnant, pregnant women or lactating women or women who desire to become pregnant.
2. Urological problems	
  1) Have previous history of urinary infection (e.g., bacterial cystitis, bladder tuberculosis, urethritis, genital chlamydia infection, and genital herpes) within 12 weeks;
  2) Currently diagnosed with any of following diseases, and/or current urinary symptoms (i.e., bladder pain, bladder discomfort, urinary frequency, persistent urge to urinate, and/or urinary urgency) are caused primarily by these diseases:
       a. Bladder diseases (overactive bladder, neurogenic bladder, bladder stone, radiation cystitis)
       b. Urethral diseases (urethral diverticulum, urethral stricture, urethral stone)
       c. Gynaecological diseases (endometriosis, uterine fibroids, vaginitis, menopausal syndrome, pelvic organ prolapse)
       d. Others (neurogenic urinary frequency, polyuria)
  3) Have previous history of augmentation cystoplasty or cystectomy;
  4) Have previous history of chemical compound (such as cyclophosphamide) derived cystitis.
3. Treatment related	
  1) Have history of the following therapies within 24 weeks: Hydrodistension, intravesical laser therapy, intravesical electrical coagulation, transurethral resection, pelvic reconstructive surgery, nerve block or spinal cord stimulation for pain relief;
  2) Received intravesical instillation of any drugs within 12 weeks.

Female patients with pure stress urinary incontinence (SUI) but stable bladder function admitted for anti-incontinence surgery were offered enrolment as controls.

135,091 CD45+ immune cells from bladders of 15 female patients with IC and 9 controls were captured to perform scRNA-seq to identify the specific immune cell types. Mass cytometry was performed to confirm the identified cell subsets. Then, immunofluorescence, ELISA tests, and the virus detection were performed to validate the possible biomarkers. Lastly, by integrating the results of scRNA-seq with ST, the identified immune subpopulations were re-located in the anatomical structure of IC bladders.

22 immune subpopulations were identified in the constructed landscape. Among them, macrophages, conventional dendritic cells, and effector memory CD4+ T cells had the most communications with other immune cells. Then, a significant increase of central memory CD4+ T cells, regulatory T cells, GZMK+CD8+ T cells, activated B cells, un-switched memory B cells, and neutrophils, and a significant decrease of CD8+ effector T cells, Th17 cells, follicular helper T cells, switched memory B cells, transitional B cells, and macrophages were noted in IC bladders. The enrichment analysis identified a virus-related response during the dynamic change of cell proportion, furthermore, the human polyomavirus-2 was detected with a positive rate of 95% in urine of patients with IC. By integrating the results of scRNA-seq with spatial transcriptomics, we found nearly all immune subpopulations were enriched in the urothelial region or located close to fibroblasts in IC bladders, but they were discovered around urothelium and smooth muscle cells in control bladders.

1. An immune landscape in IC bladders was constructed.
2. The interactions within immnue cells were investigated.
3. Although this study evidenced that inflammation or immunity had an important role in IC progress, it seemed that it was more likely to be a downstream manifestation after the destruction of epithelial barrier.

We constructed the immune landscape of bladder in women with IC, and then confirmed the characteristics of these immune cell subsets and elaborated the relation and interaction within them. This study sets a precedent for investigating the immune atlas for IC. The immune landscape may provide profound insight into the pathophysiology of IC and work as the foundation for the diagnosis and treatment of this disease in the future.

Andersen, A. V. et al. Long-term experience with surgical treatment of selected patients with bladder pain syndrome/interstitial cystitis. Scandinavian journal of urology and nephrology 46, 284-289, doi:10.3109/00365599.2012.669789 (2012).
Parsons, C. L. The role of a leaky epithelium and potassium in the generation of bladder symptoms in interstitial cystitis/overactive bladder, urethral syndrome, prostatitis and gynaecological chronic pelvic pain. BJU international 107, 370-375, doi:10.1111/j.1464-410X.2010.09843.x (2011).
Moncada, R. et al. Integrating microarray-based spatial transcriptomics and single-cell RNA-seq reveals tissue architecture in pancreatic ductal adenocarcinomas. Nature biotechnology 38, 333-342, doi:10.1038/s41587-019-0392-8 (2020).

Continence 2S2 (2022) 100315
DOI: 10.1016/j.cont.2022.100315