Autoimmunity to urothelial antigen causes bladder inflammation, pelvic pain, and voiding dysfunction: a novel animal model for Hunner-type interstitial cystitis

Abstract

Recent evidence revealed that Hunner-type interstitial cystitis (HIC) is a robust inflammatory disease potentially associated with enhanced immune responses and histologically characterized by epithelial denudation and lymphoplasmacytic infiltration with frequent clonal expansion of infiltrating B cells. To date, few animal models that reproduce the histological and clinical correlates of HIC have yet been established. In the present study, we aimed to develop a novel animal model for HIC via autoimmunity to the bladder urothelium using the transgenic mouse model (URO-OVA) that expresses the membrane form of the model antigen ovalbumin (OVA) as a self-antigen on the bladder urothelium. OVA-specific lymphocytes (splenocytes) were generated by immunization of C57BL/6 mice with OVA protein and injected intravenously into URO-OVA mice. The splenocytes from OVA-immunized C57BL/6 mice showed increased interferon (IFN)-γ production in response to OVA stimulation in vitro. URO-OVA mice adoptively transferred with OVA-primed splenocytes developed cystitis exhibiting histological chronic inflammatory changes such as remarkable mononuclear cell infiltration predominantly composed of T and B lymphocytes, increased vascularity, and mucosal hyperemia in the bladder at days 7–28 with a peak at day 21 tested. No systemic inflammation was found in cystitis-induced URO-OVA mice, nor was any inflammation found in wild-type C57BL/6 mice adoptively transferred with OVA-primed splenocytes. Along with bladder inflammation, URO-OVA mice demonstrated significantly increased pelvic nociceptive responses, voiding dysfunction, and upregulated mRNA expression levels for IFN-γ, tumor necrosis factor-α (TNF-α), and substance P precursor in the bladder. This model reproduces the histological and clinical features of human HIC, providing a novel model for HIC research.

INTRODUCTION

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition of unknown etiology characterized by persistent pain perceived to be related to the urinary bladder and lower urinary tract symptoms such as urinary frequency and urgency (1, 2). Recently, crucial concerns over this debilitating condition has been clinically and socially heightened, as it severely affects patients’ quality of life but lacks promising treatments due to the unknown pathophysiology (3). IC/BPS comprises a symptom syndrome complex and presents with a wide variety of clinical phenotypes with potentially different etiologies. To date, the definition, diagnosis, and classification of IC/BPS remain to be established due to the unclear syndrome landscape, diverse sites and degrees of clinical symptoms, and varying histology. However, IC/BPS with Hunner lesions [Hunner-type IC (HIC)] has recently emerged as a well-recognized clinically relevant phenotype, that is characterized by the cystoscopic presence of Hunner lesions, the disease hallmarking reddish mucosal lesions (4, 5). Growing evidence has shown that this subtype is a distinct immunological inflammatory disease entity with proven bladder pathology (6–8). We recently further indicated that frequent clonal expansion of infiltrated B cells and urothelial denudation are the characteristic histological features of the HIC bladder (9). In addition, a number of early studies have reported high incidence and titers of autoantibodies as well as antiurothelial autoantibodies in the serum and/or bladder of patients with IC (10–14). Collectively, all these findings suggest that autoimmunity to the bladder urothelium may underlie the pathophysiology of HIC. However, few animal models that reproduce the histological and clinical features of HIC have yet been established. Hence, in this study, we aimed to develop a novel animal model that mimics the histological and clinical features by inducing autoimmunity to urothelial antigen using a transgenic mouse model (URO-OVA) that expresses the membrane form of the model antigen ovalbumin (OVA) as a self-antigen on the urothelium (15).

Herein, we report that adoptive transfer of OVA-primed lymphocytes from C57BL/6 mice to URO-OVA mice caused cystitis exhibiting histological chronic inflammatory changes such as remarkable mononuclear cell infiltration predominantly composed of T and B lymphocytes, increased vascularity, and mucosal hyperemia, in conjunction with increased pelvic nociceptive responses and voiding dysfunction, mimicking the histological and clinical features of HIC.

MATERIALS AND METHODS

Ethics Statement

All animal procedures in the present study were approved by University of Iowa Animal Care and Use Committee (Permit Number: 1308153) and conducted according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Mice

The URO-OVA mice were previously developed in our laboratory and were of the C57BL/6 genetic background (15). C57BL/6 mice were obtained from Charles River (Wilmington, MA). All mice were housed in a pathogen-free facility at the University of Iowa Animal Care Facility under conditions of constant humidity (30%–70%) and temperature (20°C–26°C) with a 12:12-h light-dark cycle. Mice were provided with irradiated Envigo Teklad laboratory diet no. 7913 (NIH-31 Modified Open Formula Mouse/Rat sterilizable diet) and filtered water from the Edstrom automatic watering system. Euthanasia was performed by exposing the animals to 100% CO₂ at a flow rate of 4 L/min until 1 min after breathing stopped.

Generation of Antigen OVA-Specific Lymphocytes

C57BL/6 mice (female, 6–8 wk old) were immunized by subcutaneous injection of 100 µg of OVA (Sigma-Aldrich, St. Louis, MO) emulsified with complete Freund’s adjuvant (CFA, Sigma-Aldrich). At 14 days later, the OVA-immunized mice were euthanized, and the spleens were removed under sterile conditions, minced, filtered through a fine nylon mesh, placed in ACK lysing buffer (0.15 M NH₄Cl, 1.0 mM KHCO₃, and 0.1 mM Na₂EDTA, pH 7.4) to remove red blood cells, washed twice with RPMI-1640 medium, and then subjected to Ficoll–Paque gradient centrifugation to fractionate splenocytes. Viability of the fractionated splenocytes by trypan blue exclusion exceeded 90%. The splenocytes were prepared at a concentration of 4.6 × 10⁸ cells/mL in PBS for adoptive transfer injection to URO-OVA mice or cultured at 2 × 10⁶ cells/well in complete culture medium for in vitro cytokine production analysis.

In Vitro Analysis of Cytokine Production by OVA-Primed Splenocytes

The fractionated splenocytes from OVA-immunized C57BL/6 mice were resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin, seeded in a 48-well plate at a density of 2 × 10⁶ cells/well and cultured in the presence or absence of OVA immunogen (10 µg/mL) at 37°C in a 5% CO₂ humidified incubator for 3 days. The production of interferon (IFN)-γ in culture supernatants was then evaluated using R&D Systems DuoSet mouse enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Adoptive Transfer of OVA-Primed Splenocytes in URO-OVA and Wild-Type C57BL/6 Mice

URO-OVA mice (female, 6–8 wk old) were intravenously injected via the orbital sinus with OVA-primed splenocytes (4.6 × 10⁷ cells in 100 µL PBS per mouse) under anesthesia with isoflurane (1%–3% in oxygen) by mask. At days 7, 14, 21, and 28 after the adoptive transfer injection, mice were analyzed for phenotypic and functional changes including bladder histology, bladder gene expression levels, pelvic nociceptive responses, and voiding behavior. At each timepoint, bladders were collected immediately after pelvic nociceptive and voiding behavior analyses, cut in half, and stored in −80°C for subsequent histological and gene expression examinations. Sex- and age-matched normal URO-OVA mice that did not receive OVA-primed splenocytes served as controls. Each timepoint contained three to six mice for both cystitis and control groups. In a separate experiment, URO-OVA and wild-type C57BL/6 mice (both female, 8 wk old and 5 mice per group) were intravenously injected with OVA-primed splenocytes as described above. At day 21 after the adoptive transfer injection, the bladder, lung, liver, spleen, and kidney were collected and processed for histological analysis.

Bladder and Other Organ Tissue Histology

Bladders were processed for standard paraformaldehyde fixation, paraffin embedment, and section preparation for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) according to the routine protocols of our facility as previously described (15). In H&E-stained slides, stromal inflammation was evaluated based on the degrees of inflammatory cell infiltration and stromal inflammatory changes such as edema and vascularity (grade 0: none or minimal inflammatory cell infiltration with few stromal changes; grade 1: mild infiltration with mild stromal changes; grade 2: moderate infiltration with moderate stromal changes; and grade 3: moderate-to-severe infiltration with severe stromal changes). The bladder histological assessment was performed in a blinded manner by a surgical pathologist (D.M.). The lung, liver, spleen, and kidney were similarly stained with H&E solution and assessed for histological analysis.

Bladder Immunohistochemistry

For bladders that showed histological evidence of inflammation, we performed subsequent immunohistochemical characterization of infiltrating mononuclear cells. Antigen retrieval was performed by microwave treatment with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 min, followed by incubation with G-Block (Genostaff, Tokyo, Japan) and avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA). The sections were incubated with primary antibodies at 4°C overnight. We used rabbit anti-mouse CD3ɛ (0.2 µg/mL, clone D4V8L, 99940, Cell Signaling Technologies, Danvers, MA) and CD19 (0.1 µg/mL, clone D4V4B, 90176, Cell Signaling Technologies) monoclonal antibodies to detect T and B lymphocytes, respectively. The sections were then incubated with biotin-conjugated anti-rabbit secondary antibody (Dako, Glostrup, Denmark) for 30 min at room temperature, followed by addition of peroxidase conjugated streptavidin (Nichirei Bioscience, Tokyo, Japan) for 5 min. Peroxidase activity was visualized by diaminobenzidine. The sections were counterstained with Mayer’s hematoxylin (MUTO, Tokyo, Japan), dehydrated, and then mounted in Malinol (MUTO, Tokyo, Japan). An appropriate isotype-matched control rabbit IgG (X0936, Dako, Glostrup, Denmark) for CD3 and CD19 antibodies was included (Supplemental Fig. 1; all Supplemental Material is available at https://doi.org/10.6084/m9.figshare.12489539.v1). Degrees of the infiltration of T and B lymphocytes were assessed in a similar manner to that of inflammatory cell infiltration in H&E-stained slides.

Quantitative Assessment of Pelvic Nociception

As previously described (16, 17), mice were kept in individual Plexiglas chambers (6 × 10 × 12 cm) with a stainless-steel wire grid floor and allowed to acclimate for 60 min before testing. Sensory threshold of pelvic nociception was measured using electronic von Frey anesthesiometer (VFA) (ITC Inc. Life Science, Woodland Hills, CA) (18). A semi-rigid filament of the evaluator was advanced perpendicularly to the skin of the lower abdominal area, in general vicinity of the bladder, until the subject was seen to show a positive response defined as sharp abdominal retraction, brisk licking or scratching of the stimulated area, or jumping. The maximum force applied by the VFA filament at the time the positive behavioral response occurred was recorded as the measured sensory threshold. In total, five trials were completed for each subject, allowing 30-s intervals between measurements. In the five threshold values obtained for each subject, the highest and lowest values were excluded and the three middle values were averaged to assign each subject a sensory threshold (19).

Voiding Behavior Analysis

Voiding behaviors of mice were evaluated using micturition cages (Columbus Instruments, Columbus, OH) for the 24-h real-time urine outputs with 12-h light and 12-h dark cycles, as described previously (20–22). Mice were kept in individual cages and had free access to drinking water but were restrained from solid food to prevent feces from interfering with the measurement of urine outputs. Urinary frequency, voided volume per micturition, and total urine volume were automatically recorded using Oxymax software (Columbus Instruments, Columbus, OH).

RT-PCR

Total RNAs were extracted from the bladder using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA). cDNAs were synthesized using Invitrogen SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) and Oligo dT as previously described (17, 22). Polymerase chain reaction (PCR) amplification was performed on cDNA products using Taq DNA polymerase (New England Biolabs, Ipswich, MA) and the following sequence-specific primer pairs: 5′-TGAACGCTACACACTGCATCT-3′ and 5′-GACTCCTTTTCCGCTTCCTGA-3′ (459 bp) for IFN-γ, 5′-CGTCAGCCGATTTGCTATCT-3′ and 5′-CGGACTCCGCAAAGTCTAAG-3′ (206 bp) for tumor necrosis factor (TNF)-α, 5′-ATGACTCTGCGTTTGCCCT-3′ and 5′-TTGGAGCTGCCCACAATGA-3′ (280 bp) for tachykinin-1 [substance P precursor (pre-SP)], and 5′-GTTCCAGTATGACTCCACT-3′ and 5′-GTGCAGGATGCATTGCTG-3′ (321 bp) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Based on the previous PCR kinetics in our laboratory, GAPDH was amplified for 25 cycles, and other molecules were amplified for 40 cycles. The PCR products were run on 1% agarose gels, stained with ethidium bromide, and imaged by a Gel Doc EZ Imager (Bio-Rad Laboratories, Hercules, CA). The PCR band density of each gene was quantified by digital image analysis using ImageJ software (US National Institutes of Health: http://rsb.info.nih.gov/ij) and normalized to that of GAPDH from the same sample.

Statistical Analysis

Data were compared using the Wilcoxon rank-sum test for two-group comparison or the Steel–Dwass test for multiple groups. All statistical analyses were performed using GraphPad Prism software, v. 8 (GraphPad Software, San Diego, CA) and JMP software, v. 14 (SAS Institute, Cary, NC). P values <0.05 were considered statistically significant.

RESULTS

Adoptive Transfer of OVA-Primed Splenocytes From C57BL/6 Mice Caused Bladder Autoimmune Inflammation in URO-OVA Mice

To generate OVA-specific T and B lymphocytes for the induction of autoimmune cystitis in URO-OVA mice, we immunized C57BL/6 mice with CFA-emulsified OVA protein. At day 14 after immunization, splenocytes were prepared and intravenously injected into URO-OVA mice. A portion of the splenocytes was cultured in the presence or absence of the OVA immunogen for 3 days to validate the OVA immunization. We observed increased IFN-γ production by the splenocytes stimulated with OVA (Fig. 1), indicating that the immunization induced OVA-specific immune cells in C57BL/6 mice. After adoptive transfer of OVA-primed splenocytes, URO-OVA mice developed histological bladder inflammation at days 7–28 with a peak at day 21 tested (Fig. 2 and Table 1). The inflamed bladders exhibited remarkable mononuclear cell infiltration, increased vascularity, mucosal hyperemia, and interstitial edema (Fig. 2, A and C). Immunohistochemical detection further revealed that the infiltrating mononuclear cells were predominantly composed of T (CD3⁺) and B (CD19⁺) lymphocytes (Fig. 2A). Dense T and B lymphocytes were observed in the subepithelial layer (Fig. 2A). Some T lymphocytes but not B lymphocytes were found in the epithelial layer (Fig. 2, A and B). However, despite these T and B lymphocyte infiltrations, there was no clear urothelial denudation observed in the inflamed bladders. Sex- and age-matched normal URO-OVA mice showed no or few histological changes in the bladder over the course of all timepoints (Fig. 2, A and C, and Table 1), and thereby immunohistochemistry was not performed for these control mice. To determine if the induction of bladder inflammation in URO-OVA mice was urothelial OVA-antigen specific, in a separate experiment both URO-OVA and wild-type C57BL/6 mice were adoptively transferred with OVA-primed splenocytes. At day 21 after the cell transfer, the bladder, lung, liver, spleen, and kidney were collected and processed for histological analysis. Again, we observed the bladder inflammation in URO-OVA mice adoptively transferred with OVA-primed splenocytes (Fig. 3). No inflammation was observed in the other organs in cystitis-induced URO-OVA mice, nor was any inflammation observed in all organs tested, including the bladder in wild-type C57BL/6 mice adoptively transferred with OVA-primed splenocytes (Fig. 3). Along with the bladder histological changes, the inflamed bladders of URO-OVA mice expressed significantly increased levels of mRNAs for IFN-γ (days 7–21), TNF-α (days 7–21), and pre-SP (days 7–14) compared with the bladders of sex- and age-matched normal URO-OVA mice (Fig. 4). These results indicate that adoptive immunity to the urothelial OVA antigen caused autoimmune cystitis with characteristics of T and B cell infiltration, increased vascularity and mucosal hyperemia in URO-OVA mice, mimicking the histological features of HIC.

Figure 1.

Splenocytes from ovalbumin (OVA)-immunized C57BL/6 mice produce interferon (IFN)-γ in response to OVA stimulation in vitro. C57BL/6 mice (n = 25) were immunized by subcutaneous injection of 100 µg of OVA emulsified with complete Freund’s adjuvant (CFA) at day 0 and euthanized at day 14. Splenocytes were prepared, pooled, and cultured in the absence or presence of the OVA immunogen at 10 µg/mL for 3 days, followed by ELISA analysis of IFN-γ in culture supernatants. Data are shown as means ± SD, from duplicate determinations. P < 0.01 compared with nonstimulated splenocytes.

Figure 2.

Adoptive transfer of splenocytes from ovalbumin (OVA)-immunized C57BL/6 mice induces histological bladder inflammation in urothelium-ovalbumin (URO-OVA) mice. A: URO-OVA mice developed bladder inflammation at day 7, which perpetuated to day 28 with a peak at day 21, as manifested by histological hematoxylin and eosin (H&E) staining. Immunohistochemistry (IHC) analysis revealed remarkable infiltration of CD3-positive T and CD19-positive B lymphocytes in the inflamed bladders over the course of the experiment. Infiltrating T lymphocytes in the bladder epithelium are indicated by arrows. Increased vascularity, mucosal hyperemia, and interstitial edema are indicated by asterisks, arrowheads, and horizontal double asterisks, respectively. No histological changes were observed in the bladders of sex- and age-matched normal URO-OVA mice. Lower three panels (magnification, ×400) at each timepoint were the enlarged images of the areas outlined by the black-line rectangular boxes in the corresponding upper images (magnification, ×150). Results are representative of five cystitis-induced mice and six normal mice for all four timepoints. B: cystitis-induced URO-OVA mice (day 21) showed CD3-positive T lymphocyte infiltration into the bladder epithelium (arrows). No CD19-positive B lymphocyte infiltration was found in the bladder epithelium. These panels are the enlarged images (magnification, ×300) of the areas outlined by the red-line rectangular boxes in (A). Results are representative of five cystitis-induced mice at day 21. C: the bladders of cystitis-induced URO-OVA mice (day 21) showed dense mononuclear cell infiltration (arrows), increased vascularity (asterisks), mucosal hyperemia (arrowheads), and interstitial edema (horizontal double asterisks) (magnification, ×300). No such changes were observed in the bladders of sex- and age-matched normal URO-OVA mice. Results are representative of five cystitis-induced mice at day 21 and six normal mice.

Table 1.

Bladder inflammatory assessment

	Day 7				Day 14				Day 21				Day 28
	Grade 0	Grade 1	Grade 2	Grade 3	Grade 0	Grade 1	Grade 2	Grade 3	Grade 0	Grade 1	Grade 2	Grade 3	Grade 0	Grade 1	Grade 2	Grade 3
Cystitis (n = 5)
Inflammation (hematoxylin and eosin)	0	1	3	1	0	1	2	2	0	1	1	3	0	1	3	1
T lymphocytes (CD3)	1	2	1	1	1	1	2	1	0	1	2	2	0	2	2	1
B lymphocytes (CD19)	1	4	0	0	1	3	1	0	0	2	3	0	0	2	3	0
Normal (n = 6)
Inflammation (hematoxylin and eosin)	5	1	0	0	5	1	0	0	6	0	0	0	5	1	0	0
T lymphocytes (CD3)	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE
B lymphocytes (CD19)	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE	NE

Grade 0, none or minimal; grade 1, mild; grade 2, moderate; grade 3, moderate-to-severe; NE, not evaluated.

Figure 3.

Adoptive transfer of ovalbumin (OVA)-primed splenocytes induces bladder inflammation in urothelium-ovalbumin (URO-OVA) mice but not in wild-type C57BL/6 mice. Cystitis-induced URO-OVA mice at day 21 (top) showed histological inflammation in the bladder (magnifications, ×40 and ×400) but not in the lung, liver, spleen, and kidney (magnification, ×100). Sex- and age-matched wild-type C57BL/6 mice adoptively transferred with the same OVA-primed splenocytes at day 21 (bottom) showed no inflammation in the bladder (magnifications, ×40 and ×400) and the other organs tested (magnification, ×100). Results are representative of five URO-OVA mice and five C57BL/6 mice.

Figure 4.

Cystitis-induced urothelium-ovalbumin (URO-OVA) mice express increased levels of mRNAs for interferon (IFN)-γ, TNF-α, and substance P precursor (SP) precursor in the bladder. A: A representative electrophoresis image of RT-PCR products from the bladders of normal and cystitis-induced URO-OVA mice. The inflamed bladders (day 7) expressed substantially increased levels of mRNAs for IFN-γ, TNF-α, and pre-SP compared with the sex- and age-matched normal bladders. GAPDH was used as an internal control. Three bladders for each group are shown. M, a 100-bp DNA ladder. The image was cropped from three different gels run and exposed in the same experimental conditions. The noncontiguous lanes are indicated by white spaces. B: the time-course of mRNA expression in the bladders of normal and cystitis-induced URO-OVA mice. The inflamed bladders expressed significantly higher levels of mRNAs for IFN-γ, TNF-α, and pre-SP at days 7, 14, and 21 after cystitis induction compared with the sex- and age-matched normal bladders. The PCR bands were quantified by densitometry and normalized to those of GAPDH from the same samples. Three bladders for each group were analyzed. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the normal bladders.

Bladder Autoimmune Inflammation Caused by Adoptive Immunity Is Associated With Increased Pelvic Nociception and Voiding Dysfunction in URO-OVA Mice

Along with the development of autoimmune cystitis, URO-OVA mice exhibited significantly decreased sensory thresholds of pelvic nociception at days 7–28 after cystitis induction (Fig. 5). In addition, the cystitis-induced URO-OVA mice exhibited a clear trend of increase in urinary frequency over the course of all timepoints with significant changes at days 7–21 after cystitis induction compared with sex- and age-matched normal URO-OVA mice (Fig. 6A). In parallel, the cystitis-induced URO-OVA mice also showed a clear trend of decrease in mean voided volume over the course of all timepoints with significant changes at days 14–28 after cystitis induction compared with sex- and age-matched normal URO-OVA mice (Fig. 6D). All these voiding behavior changes were observed during both light and dark periods (Fig. 6, B, C, E, and F). These results indicate that adoptive immunity-caused autoimmune cystitis is associated with increased pelvic nociception and voiding dysfunction in URO-OVA mice, mimicking the clinical features of HIC.

Figure 5.

Cystitis-induced urothelium-ovalbumin (URO-OVA) mice show significantly decreased sensory thresholds of pelvic nociceptive response. URO-OVA mice (n = 5 per group) were evaluated for pelvic nociceptive responses at days 7, 14, 21, and 28 after cystitis induction using electric von Frey anesthesiometer (VFA). Sex- and age-matched normal URO-OVA mice (n = 6 per group) were included for comparison. Data are shown as means ± SE, gram of sensory threshold. *P < 0.01 and **P < 0.001 compared with the normal groups.

Figure 6.

Cystitis-induced URO-OVA mice show significant changes in voiding habits. URO-OVA mice (n = 5 mice per group) were evaluated for voiding habits at days 7, 14, 21, and 28 after cystitis induction using computer-interfaced micturition cages. Sex- and age-matched normal URO-OVA mice (n = 6 per group) were included for comparison. Voiding frequency and mean voided volume during the 24-h (A, D), light (B, E), and dark (C, F) periods were measured. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the normal groups.

DISCUSSION

In the present study, we demonstrated that adoptive immunity to the urothelial antigen induces bladder chronic inflammation characterized by subepithelial remarkable inflammatory cell infiltration, neovascularization, and hyperemia, in conjunction with increased pelvic nociception and voiding dysfunction, representing the histological and clinical features of human HIC. The cystitis induction in URO-OVA mice was urothelial OVA-antigen specific, as no systemic inflammation was found in the cystitis-induced URO-OVA mice, nor was any inflammation found in wild-type C57BL/6 mice adoptively transferred with OVA-primed splenocytes. Of note, to the best of our knowledge, we showed for the first time the evidence of robust B cell infiltration in the bladder tissues of mice with autoimmune cystitis by immunohistochemistry, which is unveiled to be a key inflammatory feature of HIC and has never been shown in any of the past cystitis murine models.

Despite of the enormous effort to understand the mechanism of the disease, the etiology of IC/BPS remains elusive and thereby no animal models that properly reproduce the characteristics of IC/BPS have yet been developed, resulting in the lack of promising treatments for the human disease (23, 24). Today, IC/BPS is one of the most challenging diseases in urology. However, recent evidence sheds light on the landscape of IC/BPS (4). IC/BPS can be, at least, categorized into the two entities with respect to cystoscopic, histological, genomic, and clinical characteristics: HIC and other forms that lack Hunner lesions (6, 25, 26). Prior studies revealed that HIC is truly an inflammatory disease totally distinct from IC/BPS without Hunner lesions, which is a noninflammatory disorder with few bladder pathological changes and potentially associated with neurophysiological dysfunction (6, 27–29). Our recent RNA sequencing analysis showed that genes and biological pathways related to immunological inflammatory processes such as T and B receptor signaling pathways, antigen processing and presentation pathways, T helper (Th)1 and Th2 cell differentiation pathways, and natural killer cell-mediated cytotoxicity pathway were significantly upregulated in HIC (26). In addition, histological evidence of deposits of immunoglobulin and complement in the bladder tissues of patients with HIC has been reported in the past (12). In line with this observation, we recently found predominant B cell infiltration, frequent B cell clonal expansion, and increased chemokine (C-X-C motif) receptor 3-positive B cells in the HIC bladder (7, 9). Taken together, all these observations strongly suggest that B cell abnormalities were the novel, crucial characteristic features of HIC. Since the early 1990s, researchers have developed several autoimmune cystitis animal models through targeting the urothelium or bladder tissues (6, 30–32). However, none of them has shown the histological evidence of extensive B cell infiltration in the bladder tissue, which is revealed to be an important inflammatory property of HIC as mentioned in the INTRODUCTION. Our present model for the first time clearly demonstrated the robust B cell infiltration, along with T cell infiltration, in the bladder of URO-OVA mice, reproducing the histological feature of human HIC.

Another cardinal histological feature of HIC is urothelial denudation. The urothelium is frequently detached thoroughly at the sites of Hunner lesions. Notably, these histological changes (chronic inflammatory changes, B cell abnormalities, and urothelial denudation) are not confined to Hunner lesions but can be seen even in the entire bladder, implying the pancystitic nature of HIC inflammation (9, 25). In clinical aspects, the female preponderance and the high prevalence of comorbid systemic autoimmune diseases are well-known epidemiological features of IC/BPS (33–35). It is also known that patients with systemic autoimmune diseases such as Sjogren’s syndrome, systemic lupus erythematosus, and autoimmune thyroiditis are frequently afflicted with coexisting bladder disorders, which have histological evidence of deposits of immunoglobulin and complement in the bladder tissues and manifest lower urinary tract symptoms similar to patients with HIC (36–38). High incidence and titers of autoantibodies in the serum and bladder of patients with IC have been previously reported (10, 14). Interestingly, antiurothelial autoantibodies in patients with IC have also been reported in the past (11–13). Taken together, these clinicopathological evidences strongly suggest the potential autoimmune nature of HIC and the importance of autoimmunity to the bladder urothelium in the pathophysiology of HIC. Although our model developed urothelial antigen (OVA)-mediated autoimmune cystitis and exhibited remarkable bladder infiltration of T and B lymphocytes, it did not display urothelial denudation. This observation suggests that urothelial denudation seen in human HIC may not result from the primary autoimmune responses to the urothelial components but rather from more complicated mechanisms involving specific immunological inflammatory reactions underlying the pathophysiology of HIC (6). However, it is possible that the lack of urothelial denudation in the animal model was due to the insufficient experimental period (i.e., 28 days) in the present study. Another possible reason for the lack of urothelial denudation could be due to the use of splenocytes from C57BL/6 mice immunized with one dose of OVA for cystitis induction in URO-OVA mice. This one-dose OVA immunization might induce only suboptimal OVA-specific T lymphocytes and no or minimal OVA-specific B lymphocytes in C57BL/6 mice. In support of this assumption, we observed that some T lymphocytes but not B lymphocytes infiltrated into the bladder epithelium where OVA antigen is expressed in cystitis-induced URO-OVA mice.

The present study has several limitations. First, like the most previously developed autoimmune cystitis murine models, we failed to observe the urothelial denudation as explained above. Second, only female mice were used due to the female predominance of patients with HIC (39). Conflicts of mouse estrous cycle with the study results were not considered. Third, we did not evaluate whether the infiltrating T and B lymphocytes in the bladder of cystitis-induced URO-OVA mice were truly derived from the transferred OVA-primed splenocytes. To further characterize this animal model, we will explore the pathological mechanisms of the autoimmune cystitis by labelling the OVA-primed splenocytes for cystitis induction in URO-OVA mice, determine if urothelial denudation is a late event of the autoimmune cystitis by extending the experimental period through repeated adoptive transfers of OVA-primed splenocytes in URO-OVA mice, perform multiple OVA immunizations in C57BL/6 mice to achieve both optimal OVA-specific T and B lymphocytes for cystitis induction in URO-OVA mice, and identify the role of B cells in the autoimmune cystitis by using URO-OVA mice genetically deficient in B cells. In summary, we hereby show that adoptive immunity to the urothelial antigen causes chronic bladder inflammation characterized by predominant T and B cell infiltration, increases pelvic nociceptive responses, and voids dysfunction in URO-OVA mice, mimicking the histological and clinical features of human HIC. Our study provides a novel animal model closest to the human HIC conditions and a basic research platform for future development of effective treatments for HIC.

GRANTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01DK111396 (to Y.L.), KAKENHI Grants-in-Aid 19K 18552 from the Japanese Society for the Promotion of Science (to Y.A.), and Takara Bio Inc (to D.M.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Y.A. and Y.L. conceived and designed research; Y.A., J.-R.Y., and D.L. performed experiments; Y.A. and Y.L. analyzed data; Y.A. and Y.L. interpreted results of experiments; Y.A., J.-R.Y., and Y.L. prepared figures; Y.A. and Y.L. drafted manuscript; K.J.K., M.A.O., S.K.L., D.M., H.K., Y.H., and Y.L. edited and revised manuscript; Y.A., J.-R.Y., K.J.K., M.A.O., S.K.L., D.L., D.M., H.K., Y.H., and Y.L. approved final version of manuscript.