Neuroinflammatory gene expression analysis reveals potential novel mediators and treatment targets in interstitial cystitis with Hunner lesions

Introduction

Interstitial cystitis (IC) is characterized by pelvic pain, pressure, or discomfort related to the urinary bladder, often accompanied by urinary urgency or frequency, and relieved by voiding (1). IC is often debilitating, its etiology remains enigmatic, and it affects 2.7–6.5% of the U.S. population (2,3). Patients with IC incur about $7,100 in annual healthcare costs, nearly double the annual healthcare costs than patients without the condition (4). IC is a heterogenous condition, with no one-size-fits-all treatment, but rather treatment tailored to the individual. Commonly, patients are treated with a multimodal regimen of progressively escalated therapies, before they reach acceptable symptomatic control (5). Men are subject to misdiagnoses including prostatitis, benign prostatic enlargement, and epididymitis, and thus provide unique challenges practitioners (6). Due to this, along with higher prevalence of IC in women, few studies have focused on the condition in men. As yet there are no reliable and validated biomarkers to guide both diagnosis and treatment in this population.

There has been recent investigation of IC biomarkers with antiproliferative factor, epidermal growth factor, heparin-binding epidermal growth factor, glycosaminoglycans, and bladder nitric oxide as some of recent focus (7). However, due to difficulty or lack of practicality of assays, such as the need to culture bladder urothelial cells for detection of antiproliferative factor activity, diagnosis remains based on symptomatology and cystoscopic findings. An important subtype of IC is those patients with Hunner lesions found on cystoscopy. These patients are often older, have more severe symptoms and are more likely to respond to immunotherapy such as cyclosporine (2,8). Nevertheless, diagnosis of Hunner lesions is inconsistent and prevalence can vary widely depending on the Urologist’s experience identifying them. Therefore, identification of novel objective biomarkers would be helpful to guide effective therapy.

The NanoString nCounter is a novel rapid and automated platform allowing for direct mRNA measurement for up to 800 genes, using a small sample without the need for cDNA or polymerase chain reaction (9). We sought to compare RNA profiles of patients with IC with Hunner lesions relative to healthy controls. We utilized a pre-designed cassette with nearly 800 genes associated with inflammation and/or neuropathic pain, and set out to study gene expression in blood, urine, and bladder tissue for this set of neuroinflammatory genes in men with IC compared to asymptomatic controls. Our approach allows for simultaneous measurement of gene interaction networks, as the orchestrated expression of interrelated genes, rather than any single gene, may play a key role in IC pathophysiology. We hypothesized that men with IC would have unique gene expression signatures associated with neuroinflammatory pathways, relative to asymptomatic controls. We present the following article in accordance with the MDAR reporting checklist (available at https://dx.doi.org/10.21037/tau-21-657).

Methods

The study was approved by the institutional IRB (#19-1515) and appropriate written consent was obtained for all subjects. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Blood, urine, and bladder biopsies were collected from 6 men with the clinical diagnosis of symptomatic IC found to have Hunner lesions (10). IC patients were excluded if they were not currently symptomatic with pain and lower urinary tract symptoms (LUTS), or if they had active infection (newly found or being treated with an antibiotic). IC patients were also excluded from our investigation if they were receiving immune-modulating treatments (e.g., corticosteroids), with the exception of cyclosporine for the secondary analysis of comparison of IC patients not on cyclosporine to the patient on cyclosporine. A single bladder biopsy was taken by an attending urologist with recognized expertise in the diagnosis and management of IC, from a visible Hunner lesion. The biopsy was obtained during cystoscopy under general anesthesia using cold cup biopsy forceps prior to lesion fulguration. At least 20 mL urine and approximately 3 mL blood were obtained either during a clinic visit or on the day of surgery prior to starting antibiotics or anesthesia. Urine and blood were similarly collected from 7 asymptomatic men, serving as controls, without signs or symptoms of UTI or LUTSs, presenting for a vasectomy. Benign biopsies were analyzed from our tissue bank, from 2 male patients with no documented history of LUTS or IC. These men underwent biopsy for suspicion of bladder cancer, and biopsies were taken from areas remote to any target lesion. Men were excluded from this group for evidence of UTI on preoperative urine culture, or other diagnosis of LUTSs or pain.

All samples were processed and frozen within 6 hours of collection. Samples were stored at the appropriate temperature (−20 °C for tempus tubes, −80 °C for urine pellets, wherein RNA is stable indefinitely) for up to 9 months before being used for analysis. Three mL of blood was collected in Tempus Blood RNA tubes (Applied Biosystems; ThermoFisher Scientific), which contain a stabilizing reagent that immediately lyses blood cells and inactivates RNases. RNA isolation and analysis were performed as previously described (11). The RNA is stable indefinitely when stored at −20 °C. RNA was isolated from the blood samples using the Tempus Spin RNA Isolation Kit.

Urine samples were centrifuged at 3,000 g for 30 minutes at 4 °C in 50 mL conical tubes. The pellets were resuspended in 1 mL PBS, transferred to 2 mL microcentrifuge tubes and centrifuged at 10,000 rpm for 4 minutes at room temperature. The PBS was removed and the urine pellets were then immediately stored at −80 °C. RNA was isolated from the urine pellets using the Qiagen Rneasy Mini Kit. RNA from blood samples was quantified using a Nanodrop ND100 spectrophotometer. The mean RNA yield was 8.53 µg (range, 3.5–13.6 µg) and the OD260/280 ratios were all >2.1. RNA from urine samples was measured both with a Nanodrop ND100 spectrophotometer to determine the OD260/280 ratios, and the Qubit4 for RNA concentration. The mean RNA yield was 291 ng (range, 0.1 to 1,600 ng) and the mean OD260/280 ratio was 1.8. A total of 100 ng RNA was used for gene expression analysis with the 770-gene NanoString Human Neuroinflammation gene panel.

Statistical analysis

Data were imported into ROSALIND^® (https://rosalind.onramp.bio/) for quality control, normalization, analysis of fold-changes and P values, and identification of enriched pathways and gene sets. Differential gene expression was considered biologically and statistically significant if there was a greater than 1.5-fold change compared to controls (the asymptomatic control group for the blood and urine samples, and the tissue bank group for the tissue sample), and Benjamini-Hochberg adjusted P value was P<0.05. Patients were clinically phenotyped with the Urinary, Psychosocial, Organ-Specific, Infection, Neurologic/Systemic and Tenderness (UPOINT) system (12). The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Results

In the IC group, mean patient age was 61.5 years (range, 44–69 years) and median symptom duration was 8 years (range, 2–17 years). Number of positive UPOINT domains ranged from 2–4 with a median of 2.5. In the control group, the mean patient age was 42.6 years (range, 30–55 years) and the patients had no reported history of LUTSs. The bladder biopsy patients were 48 and 65 years old, respectively.

Bladder

Bladder biopsy of the subjects with IC as compared to controls showed differential expression in 141 genes. The highest Nanostring gene set global significance scores were for apoptosis, adaptive immune response, and oligodendrocyte function (Table 1). The genes with the greatest significant positive fold-change were transient receptor potential cation channel, subfamily A, member 1 (TRPA1), tumor necrosis factor receptor superfamily, member 17 (TNFRSF17), lymphocyte antigen 9 (LY9) interferon regulatory factor 4 (IRF4), and chemokine (C-X-C motif) ligand 10 (CXCL10) (Table 2). The genes with the greatest significant negative fold-change were RNA binding protein, fox-1 homolog (C. elegans) 3 (RBFOX3), microtubule-associated protein tau (MAPT) (Table 2).

Urine

Urine of the subjects with IC as compared to controls showed differential expression in 123 genes. The highest gene set analysis scores were for Wnt (neuropathic pain), adaptive immune response, and angiogenesis (Table 1). The genes with the greatest significant positive fold-change were sphingosine-1-phosphate receptor 4 (S1PR4), suppressor of cytokine signaling 3 (SOCS3), colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage) (CSF2RB), neutrophil cytosolic factor 1 (NCF1), and ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2) (RAC2) (Table 3).

Blood

Blood of the subjects with IC as compared to controls showed differential expression in 41 genes. The highest gene set analysis scores were for oligodendrocyte function, carbohydrate metabolism, and matrix remodeling (Table 1). The genes with the greatest significant positive fold-change were lipocalin 2 (LCN2), CD68 molecule (CD68), granulin (GRN), interleukin 1 receptor, type II (IL1R2), and pleckstrin homology domain containing, family M (with RUN domain) member 1 (PLEKHM1) (Table 4). The genes with the greatest significant negative fold-change were radical S-adenosyl methionine domain containing 2 (RSAD2) and membrane-spanning 4-domains, subfamily A, member 1 (MS4A1) (Table 4).

Cyclosporine: bladder, urine, and blood

One of the IC patients was on cyclosporine, and thus we performed separate analyses comparing with the other IC patients. Bladder biopsy of the subject on cyclosporine as compared to those not on cyclosporine showed differential expression in 156 genes. Table 5 shows the genes with the largest difference between the patients not on cyclosporine versus the patient on cyclosporine in bladder, urine, and blood. As expected, key inflammatory genes including CD14 (501-fold in urine, 3-fold in blood), CXCL9 (248-fold in urine) and CXCL10 (ten-fold in bladder tissue) were downregulated in the patient on cyclosporine. Notably, there was no significant difference in TRPA1 expression between the two groups.

Discussion

Progress in the treatment of IC has been slow, and hampered by a lack of predictive biomarkers and limited consensus on pathophysiology. Patients with Hunner lesions are a distinct subset of IC patients with documented inflammatory lesions in the bladder that may be responsive to fulguration, direct steroid injection and cyclosporine (2,8). Our study used a novel technology to simultaneously measure the expression of neuroinflammatory genes in blood, urine and tissue between men with IC versus asymptomatic controls. While commonly known inflammatory markers were identified as expected, we found novel genes whose function could lead to new therapeutic interventions for IC. In bladder biopsies, we identified significantly different expression of 141 genes. As expected, many cytokine and chemokine genes had higher expression (e.g., CXCL10 92-fold higher, TNF 28-fold higher). Other significantly differently expressed genes included TRPA1 (1,098-fold higher) and TNFRS17 (735-fold higher). Apoptosis, adaptive immune response, and oligodendrocyte pathways had higher significance scores in bladder tissue in patients with IC relative to controls. It is thought that neurogenic inflammation of the mucosa of the bladder can cause central and peripheral nervous system changes, which may be reflected in oligodendrocyte function, and can in turn contribute to IC (13).

TRPA1 was differentially expressed at 1000-fold greater levels in IC patients relative to asymptomatic controls. TRPA1 is an ion channel that has been shown to be an important component in pain pathways (14,15). It has been implicated in inflammatory pain such as in osteoarthritis, neuropathic pain such as in diabetic neuropathy, migraine pain, and cancer pain. In addition, TRPA1 has also been shown to play an important role in “dysfunctional pain”, a category of pain with an ill-defined cause, where unexplained pain is the main presenting symptom (15). Dysfunctional pain conditions include IC, fibromyalgia, temporomandibular disorder, and irritable bowel syndrome.

TRPA1 has been shown to be associated with inflammation and LUTS in the urinary bladder (16). A gain of function mutation in TRPA1 is responsible for familial episodic pain syndrome, and interestingly, antagonists have been shown to inhibit the gain-of-function mutant channel (17). Thus, TRPA1 may be an important drug target for pain syndromes. It has been shown that TRPA1 localized to unmyelinated nerve fibers in the urothelium, suburothelium, and muscle layers of the rat urinary bladder, and that activators of TRPA1 result in increased voiding frequency, decreased voided volume, and detrusor overactivity (18). It has also been shown to localize to the urothelial and interstitial cells of the urethra, and that TRPA1 agonists can modify the tone of human urethral preparations, suggesting a role in signaling in the human urinary outflow region (19). In a rat model of IC, inhibition of the TRPA1 decreased bladder hyperactivity and pain (20). It has also been shown to be differentially expressed in bladder tissue of those with IC (21). Our results build on these findings, and importantly, have demonstrated the presence of a significant upregulation in IC patients with Hunner lesions.

S1PR4, a G protein-coupled receptor never previously demonstrated in the urine, was detected at 114-fold higher expression in the urine of IC patients than healthy controls. It is a member of the endothelial differentiation, G-protein-coupled (EDG) receptor family. It is mainly expressed in the hematopoietic system. A recent study utilizing S1PR4 knockout mice demonstrated that the main defect was in dendritic cell differentiation and cytokine secretion. In this study, dendritic cells were unable to switch T cells to Th17, and thus the immune response was shifted to a Th2 response (22). Of note, in IC, the Th17 cell-differentiation pathway has been shown to be significantly enriched in patients with IC with Hunner’s lesions (23). Patients with IC are 100 times more likely to have inflammatory bowel disease than healthy individuals (24). Interestingly, severity of colitis was significantly reduced in a S1PR4 knockout inflammatory bowel disease mouse model (22). The molecule FTY720, which has a beneficial effect in treatment of colitis was postulated to act through the synergistic inactivation of both S1PR1 and S1PR4 (25). In a Phase II randomized controlled clinical trial, Etrasimod (APD334), an S1P1, S1P4, and S1P5 receptor modulator, was more effective than placebo in terms of clinical and endoscopic improvements in patients with moderate to severe ulcerative colitis (26).

Kittaka et al. have demonstrated a potential link between TRPA1 and S1PR (27). S1P-induced responses in the dorsal root ganglia neurons in mice were partially inhibited by TRPA1 antagonists. The group used a selective TRPA1 antagonist (HC-030031), and showed that it reduced the S1P-induced increases of intracellular calcium influx. Their results suggested that calcium influx caused by S1P is mediated by TRPA1. Additional studies are needed to further understand these molecular underpinnings.

Our results, in the context of the above studies, elucidate a link between TRPA1 and S1PR in IC. TRPA1 has a 1,098-fold expression increase in bladder tissue and S1PR4 a 114-fold expression increase in urine in those with IC relative to asymptomatic controls. We propose that this combination may lead to an intracellular calcium influx and subsequent ATP release. Indeed, multiple studies have shown that ATP release in urothelial cells is controlled by a rise in intracellular calcium concentration (28,29). It is known that ATP is released from the urothelium in the context of bladder stretch. In a feline model for IC, it has been shown that swelling-evoked ATP release in IC cells was elevated relative to cells of normal cats (30). Further, bladder urothelial cells in patients with IC had augmented extracellular ATP release relative to those of healthy controls (31).

TNFRS17, also known as B-cell maturation antigen (BCMA), is preferentially expressed in mature B lymphocytes, and has been implicated in cell development and autoimmune response. Until now, it has not been detected in the bladder. TNFRS17 has been implicated in lymphomas, leukemias, and multiple myeloma, and autoimmune diseases including SLE. TNFRS17 has been shown to have inflammatory effects on myeloid cells, and to be expressed in macrophages (32). TNFRS17 has also been identified as a suitable candidate for CAR-T therapy in multiple myeloma (33).

One of our patients was on cyclosporine therapy. Although inflammatory gene expression levels were significantly lower, as expected, in blood, urine, and tissue, TRPA1 was not significantly decreased in the cyclosporine patient’s bladder tissue. This finding, in the context of our other data and the literature, supports the importance of the identification of novel inhibitors of TRPA1 for the treatment of IC. Given that TRPA1 antagonists have been developed for a variety of pain conditions, there is the possibility that therapy delivered systemically or intravesically could improve symptoms in IC, sparing the long-term toxicities of cyclosporine.

Strengths of this study include the broad range of genes tested and inclusion of a healthy control group. Limitations include the relatively small sample size, a split between control samples for fluids and tissue, an age difference between groups, and lack of histological or proteomic analysis. The small sample size reflects the rarity of IC, particularly in the male population with Hunner lesions. Indeed, the female to male ratio is 5:1, the minority of these patients harbors Hunner lesions, and there has been minimal investigation in this population (34-36). Further, cyclosporine use in the population is exceedingly rare, as it is currently recommended as a fifth line treatment for IC by the American Urological Association (37). Our study population was confined to men, because of their unique challenges and misdiagnoses, the paucity of literature on men with IC, and because they are the focus of the clinical practice. The study calls for further investigations to generalize these findings to women and other patients with IC. Future work should also address the differential genetic signatures of men with IC as compared to those with chronic prostatitis, or benign prostatic hyperplasia, as there is overlap in these conditions and their differentiation in practice can be challenging. Further, the study was not designed to assess for outcomes of IC interventions based on genetic signatures, but this will be a future focus.

In conclusion, men with IC and Hunner lesions have a diverse set of neuroinflammatory genes with differential expression compared to controls. In addition to the expected inflammatory genes, we identified genes linked to neuropathic pain through the TRP pathway and this expression was not reduced by cyclosporine. These findings open a new direction for biomarker and therapeutic discovery.

Acknowledgments

Funding: This research was funded in part by generous philanthropic support at Cleveland Clinic. Special thanks to Mr. Sam Kellett and Mr. Paul Vitek for their support of our work. Nanostring analysis was performed by the NIAID-sponsored Clinical Trials in Organ Transplantation (CTOT) NanoString Core (U01 AI063594).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://dx.doi.org/10.21037/tau-21-657

Data Sharing Statement: Available at https://dx.doi.org/10.21037/tau-21-657

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/tau-21-657). DAS serves as an unpaid editorial board member of Translational Andrology and Urology from August 2020 to July 2022. DAS reports investment interest in Triurol. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the institutional IRB (#19-1515) and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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