Cystitis-induced bladder pain is Toll-like receptor 4 dependent in a transgenic autoimmune cystitis murine model: a MAPP Research Network animal study

Abstract

Altered Toll-like receptor (TLR)4 activation has been identified in several chronic pain conditions but has not been well studied in interstitial cystitis/bladder pain syndrome (IC/BPS). Our previously published human studies indicated that patients with IC/BPS present altered systemic TLR4-mediated inflammatory responses, which were significantly correlated with reported pain severity. In the present study, we sought to determine whether altered TLR4 activation plays a role in pelvic/bladder pain seen in patients with IC/BPS using our validated IC/BPS-like transgenic autoimmune cystitis model (URO-OVA). URO-OVA mice developed responses consistent with pelvic and bladder pain after cystitis induction, which was associated with increased splenocyte production of TLR4-mediated proinflammatory cytokines IL-1β, IL-6, and TNF-α. Increased spinal expression of mRNAs for proinflammatory cytokines IL-6 and TNF-α, glial activation markers CD11b and glial fibrillary acidic protein, and endogenous TLR4 ligand high mobility group box 1 was also observed after cystitis induction. Compared with URO-OVA mice, TLR4-deficient URO-OVA mice developed significantly reduced nociceptive responses, although similar bladder inflammation and voiding dysfunction, after cystitis induction. Intravenous administration of TAK-242 (a TLR4-selective antagonist) significantly attenuated nociceptive responses in cystitis-induced URO-OVA mice, which was associated with reduced splenocyte production of TLR4-mediated IL-1β, IL-6, and TNF-α as well as reduced spinal expression of mRNAs for IL-6, TNF-α, CD11b, glial fibrillary acidic protein, and high mobility group box 1. Our results indicate that altered TLR4 activation plays a critical role in bladder nociception independent of inflammation and voiding dysfunction in the URO-OVA model, providing a potential mechanistic insight and therapeutic target for IC/BPS pain.

Keywords: Multidisciplinary Approach to the Study of Chronic Pelvic Pain; Toll-like receptor 4; cystitis; model; pain.

Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the author(s).

Figures

Fig. 1.

Toll-like receptor 4 (TLR4) plays a critical role in bladder nociception independent of inflammation in autoimmune cystitis. A: at day 7 after cystitis induction, the bladders of URO-OVA and URO-OVATLR4−/− mice were collected, sectioned, and analyzed by histological hematoxylin and eosin staining (magnification: ×40 and ×400). The normal bladders of URO-OVA and URO-OVATLR4−/− mice were included for comparison (magnification: ×40). Images are representative of 8 bladders/group. B: URO-OVATLR4−/− mice (n = 10) developed reduced pelvic nociceptive responses to von Frey filament stimulation compared with wild-type URO-OVA mice (n = 10) after cystitis induction. Normal pelvic nociceptive responses (n = 10 for each strain) were included for comparison. Data are shown as means ± SE of percent response frequency. *P < 0.05 and **P < 0.01 compared with the cystitis-induced URO-OVA group. C: URO-OVATLR4−/− mice (n = 9) developed reduced urinary bladder distention-evoked visceromotor response (VMRs) compared with wild-type URO-OVA mice (n = 6) after cystitis induction. Normal VMRs (n = 6 for each strain) were included for comparison. Data are shown as means ± SE of VMRs (in V/s). *P < 0.05 and **P < 0.01 compared with the cystitis-induced URO-OVA group.

Fig. 2.

Toll-like receptor 4 (TLR4) plays no role in voiding dysfunction in autoimmune cystitis. At day 7 after cystitis induction, both URO-OVA (left) and URO-OVATLR4−/− (right) mice were placed in micturition cages for 24-h micturition recordings (see Table 2). Baseline voiding habits were included for comparison. Data are shown as the amount (in g) of urine collected in 2-min intervals during the 24-h period. Results are representative of 5 mice for each of the two mouse strains.

Fig. 3.

Bladder nociception is associated with increased systemic and central Toll-like receptor 4 (TLR4)-mediated inflammatory responses in autoimmune cystitis. A: at day 7 after cystitis induction, splenocytes of URO-OVA mice (n = 8) were prepared and cultured in the presence of LPS at 10-fold escalating dosages ranging from 10−5 to 102 μg/ml for 24 h. Splenocytes from normal URO-OVA mice (n = 8) were included for comparison. IL-1β (top), IL-6 (middle), and TNF-α (bottom) in the conditioned culture supernatants were then evaluated by ELISA. Data are shown as means ± SE. *P < 0.05 and **P < 0.01 compared with the normal group. B: at day 7 after cystitis induction, lumbar spinal cords of URO-OVA mice were isolated and processed for total RNA extraction followed by RT-PCR analysis of IL-6, TNF-α, CD11b, glial fibrillary acidic protein (GFAP), and high mobility group box 1 (HMGB1) mRNA. GAPDH was used as an internal control. Lumbar spinal cords from normal URO-OVA mice were included for comparison. Three lumbar spinal cords for each group are shown. M, 100-bp DNA ladder. The image shown in B was cropped from six different gels run and exposed in the same experimental conditions.

Fig. 4.

Blockade of Toll-like receptor 4 (TLR4) attenuates bladder nociception in autoimmune cystitis. At day 7 after cystitis induction, URO-OVA mice were treated intravenously with vehicle or TAK-242 at 2.5 mg/kg. One hour after treatment, mice were analyzed for pelvic nociceptive responses by von Frey filament stimulation method (n = 10 mice per group; A) and bladder nociceptive responses by the urinary bladder distention-evoked visceromotor response (VMR) method (n = 9 mice for the two treated groups and n = 6 mice for the normal group; B). Data are shown as mean ± SE for both pelvic and bladder nociceptive responses. *P < 0.05 and **P < 0.01 compared with the vehicle-treated group.

Fig. 5.

Attenuated bladder nociception is associated with reduced systemic and central Toll-like receptor 4 (TLR4)-mediated inflammatory responses in autoimmune cystitis. A: at day 7 after cystitis induction, URO-OVA mice were treated intravenously with vehicle (n = 8) or TAK-242 at 2.5 mg/kg (n = 8). One hour after treatment, splenocytes were prepared and cultured in the presence of LPS at 10-fold escalating dosages ranging from 10−5 to 102 μg/ml for 24 h. Splenocytes from normal URO-OVA mice (n = 8) were included for comparison. IL-1β (top), IL-6 (middle), and TNF-α (bottom) in the conditioned culture supernatants were then evaluated by ELISA. Data are shown as means ± SE. *P < 0.05 and **P < 0.01 compared with the vehicle-treated group. B: lumbar spinal cords were isolated at 1 h after vehicle or TAK-242 treatment and processed for total RNA extraction followed by RT-PCR analysis of IL-6, TNF-α, CD11b, glial fibrillary acidic protein (GFAP), and high mobility group box 1 (HMGB1) mRNA. GAPDH was used as an internal control. Three lumbar spinal cords for each group are shown. M, 100-bp DNA ladder. The image shown in B was cropped from six different gels run and exposed in the same experimental conditions.