Inflammasomes are important mediators of cyclophosphamide-induced bladder inflammation

Abstract

Bladder inflammation (cystitis) underlies numerous bladder pathologies and is elicited by a plethora of agents such as urinary tract infections, bladder outlet obstruction, chemotherapies, and catheters. Pattern recognition receptors [Toll-like receptors (TLRs) and Nod-like receptors (NLRs)] that recognize pathogen- and/or damage-associated molecular patterns (PAMPs and/or DAMPs, respectively) are key components of the innate immune system that coordinates the production (TLRs) and maturation (NLRs) of proinflammatory IL-1β. Despite multiple studies of TLRs in the bladder, none have investigated NLRs beyond one small survey. We now demonstrate that NLRP3 and NLRC4, and their binding partners apoptosis-associated speck-like protein containing a COOH-terminal caspase recruitment domain (ASC) and NLR family apoptosis inhibitory protein (NAIP), are expressed in the bladder and localized predominantly to the urothelia. Activated NLRs form inflammasomes that activate caspase-1. Placement of a NLRP3- or NLRC4-activating PAMP or NLRP3-activating DAMPs into the lumen of the bladder stimulated caspase-1 activity. To investigate inflammasomes in vivo, we induced cystitis with cyclophosphamide (CP, 150 mg/kg ip) in the presence or absence of the inflammasome inhibitor glyburide. Glyburide completely blocked CP-induced activation of caspase-1 and the production of IL-1β at 4 h. At 24 h, glyburide reduced two markers of inflammation by 30-50% and reversed much of the inflammatory morphology. Furthermore, glyburide reversed changes in bladder physiology (cystometry) induced by CP. In conclusion, NLRs/inflammasomes are present in the bladder urothelia and respond to DAMPs and PAMPs, whereas NLRP3 inhibition blocks bladder dysfunction in the CP model. The coordinated response of NLRs and TLRs in the urothelia represents a first-line innate defense that may provide an important target for pharmacological intervention.

Keywords: bladder; caspase-1; cystitis; inflammasome; inflammation.

Fig. 1.

Detection of inflammasomes in the bladder by immunocytochemistry. Bladders from untreated rats were fixed, sectioned (5 μm), stained with the indicated antibodies, and counterstained with hematoxylin. Digital micrographs (2,560 × 1,920 pixels) were then taken at ×20 magnification. Scaled images (top) represent the entire micrograph scaled to 9.3% of the original size. Arrows indicate staining in urothelia, arrowheads indicate staining in the detrusor, and oval regions represent areas of staining in vascular elements. Areas encompassed by rectangles were cropped out of the full-sized micrographs and presented full scale in cropped images (middle). Isotype control images (bottom) were stained with the respective isotype antibodies as described in materials and methods. Micrographs were reduced to 9.3% of the original size to accurately serve as controls for the scaled images. All micrographs are from a representative experiment repeated three independent times. NLR, Nod-like receptor; ASC, apoptosis-associated speck-like protein containing a COOH-terminal caspase recruitment domain; NAIP, NLR family apoptosis inhibitory protein.

Fig. 2.

Caspase-1 activity in urothelia after intravesicular challenge with pathogen- and/or damage-associated molecular patterns (PAMPs and/or DAMPs, respectively). Bladders in unconscious rats were drained with a 28-gauge needle, and each DAMP or PAMP was instilled in the lumen (300 μl) for 2 h. Urothelial lysates were then assessed for caspase-1 activity. Treatments were as follows: lipopolysaccharide (LPS; 100 μg/ml), ATP (10 mM), monosodium urate crystals (MSU; 200 μg/ml), and flagellin (12.5 μg/ml). Bars are means ± SEM; n = 23 for control, 12 for LPS, 13 for ATP, 4 for MSU, and 6 for flagellin treatment. *Significant (P < 0.05) differences from control as assessed by ANOVA modeling.

Fig. 3.

Dosing regimen used to study the effects of glyburide (Gly) on various parameters in the cyclophosphamide (CP)-induced model of cystitis. This regimen was used for the results shown in Figs. 4–8 and Table 1. Vehicle, Gly, and CP were administered at the indicated doses, and animals were harvested for various analyses where indicated.

Fig. 4.

Effects of Gly on inflammasome activation in urothelia and urinary cytokine levels. Rats were subjected to the treatment regimen shown in Fig. 3. At the end of treatment, urine was collected, and urothelia were isolated and assayed as described in materials and methods. A: caspase-1 activity in urothelia after the indicated treatments. Bars are means ± SE; n = 5 for each group. *Significant (P < 0.001) difference from control as assessed by ANOVA modeling and Fisher's least-significant-difference (LSD) test; †significant (P < 0.002) difference from vehicle + CP treatment. B: IL-1β levels in urine after the indicated treatments normalized to creatinine. Bars are means ± SE; n = 3 for vehicle, 5 for CP, 3 for Gly, and 3 for Gly + CP treatment. *Significant (P < 0.02) difference from control as assessed by ANOVA modeling and Fisher's LSD test; †significant (P < 0.02) difference from vehicle + CP treatment. C: IL-18 levels in urine after the indicated treatments normalized to creatinine. Bars are means ± SE. No significant differences were detected.

Fig. 5.

Effect of Gly on CP-induced bladder weight changes. Rats were subjected to the treatment regimen shown in Fig. 3. At the end of treatment, bladders were harvested, cleaned of all fat, and weighed. Bars are means ± SE; n = 4 for vehicle, 8 for CP, 4 for Gly, and 7 for Gly + CP treatment. *Significant (P < 0.05) differences from control as assessed by ANOVA modeling; †significant difference from vehicle + CP treatment.

Fig. 6.

Effect of Gly on CP-induced Evan's blue dye extravasation. Rats were subjected to the treatment regimen shown in Fig. 3. At the end of the experiment, rats were injected (intravenously) with 25 mg/kg Evans blue. One hour later, rats were euthanized and their bladders were harvested A: photograph of a representative bladder from each of the indicated treatment groups. B: Evans blue extracted from the bladders treated in the indicated ways. Bladders were weighed and incubated in formamide at 56°C overnight. Absorbance at 620 nm was analyzed, and measurements (in ng) of Evan's blue were determined from a standard curve. Bars are means ± SE; n = 4 for vehicle, 4 for CP, 4 for Gly, and 7 for Gly + CP treatment. *Significant (P < 0.05) differences from control as assessed by ANOVA modeling; †significant difference from vehicle + CP treatment.

Fig. 7.

Effect of Gly on CP-induced histological changes. Rats were subjected to the treatment regimen shown in Fig. 3. At the end of the experiment, rats were euthanized, and their bladders were removed, fixed, sectioned, and then stained with hematoxylin and eosin as described in materials and methods. E, edema. The large arrow indicates urothelial ulceration (sloughing), and the small arrow indicates neutrophils. Micrographs are from a representative experiment repeated four independent times.

Fig. 8.

Effect of Gly on CP-induced urodynamics. Rats with suprapubic catheters implanted were subjected to the treatment regimen shown in Fig. 3A. At the end of the experiment, urodynamics were performed as described in materials and methods. A: representative pressure tracings from the four treatment groups. Voiding pressure, threshold pressure, intercontraction interval (ICI), and one nonvoiding contraction (NVC) are indicated. Not shown is a tracing from the scale indicating changes in weight coinciding with micturition, which is indicative of void volume.