A murine model of inflammatory bladder disease: cathelicidin peptide induced bladder inflammation and treatment with sulfated polysaccharides

Abstract

Purpose: Studies show that LL-37 is a naturally occurring urinary defensin peptide that is up-regulated during urinary tract infections. Although normal urinary LL-37 levels are antimicrobial, we propose that increased LL-37 may trigger bladder inflammation. We further suggest that anti-inflammatory sulfated polysaccharides known as semi-synthetic glycosaminoglycan ether compounds can treat/prevent LL-37 mediated bladder inflammation.

Materials and methods: C57BL/6 mice were catheterized/instilled with LL-37 (320 μM, 150 μl) for 45 minutes. Animals were sacrificed at 12 and 24 hours, and tissues were examined using hematoxylin and eosin. Separate experiments were performed for myeloperoxidase to quantify inflammation. GM-1111 semi-synthetic glycosaminoglycan ether treatments involved instillation of 10 mg/ml for 45 minutes directly before or after LL-37. Tissues were harvested at 24 hours. To compare semi-synthetic glycosaminoglycan ether efficacy, experiments were performed using 10 mg/ml heparin. Finally, tissue localization of semi-synthetic glycosaminoglycan ether was examined using a fluorescent GM-1111-Alexa Fluor® 633 conjugate.

Results: Profound bladder inflammation developed after LL-37. Greater tissue inflammation occurred after 24 hours compared to that at 12 hours. Myeloperoxidase assays revealed a 21 and 61-fold increase at 12 and 24 hours, respectively. Semi-synthetic glycosaminoglycan ether treatment after LL-37 showed mild attenuation of inflammation with myeloperoxidase 2.5-fold below that of untreated bladders. Semi-synthetic glycosaminoglycan ether treatment before LL-37 demonstrated almost complete attenuation of inflammation. Myeloperoxidase results mirrored those in controls. In heparin treated bladders minimal attenuation of inflammation occurred. Finally, instillation of GM-1111-Alexa Fluor 633 revealed urothelial coating, significant tissue penetration and binding to endovasculature.

Conclusions: We developed what is to our knowledge a new model of inflammatory bladder disease by challenge with the naturally occurring urinary peptide LL-37. We also noted that a new class of anti-inflammatory sulfated polysaccharides prevents and mitigates bladder inflammation.

Figure 1

Gross images show saline instilled control bladders harvested at 12 (A) and 24 (B) hours, and LL-37 instilled bladders harvested at 12 (C) and 24 (D) hours. Bladders were hemisected to expose inner mucosal layer. Scale bar represents 0.5 cm.

Figure 2

Histological cross section of saline instilled control bladders harvested at 12 (A) and 24 (B) hours shows no evidence of inflammation while LL-37 instilled bladders harvested at 12 (C) and 24 (D) hours show PMN infiltration (circles), microabscess (MA) formation and PMN margination out of blood vessels (rectangles). U, urothelium. SBM, submucosa. LP, lamina propria. SMC, smooth muscle cell layer. H & E, reduced from ×10. Scale bar represents 75 μM.

Figure 3

Tissue MPO inflammation quantification assay of saline control (blue bars) and LL-37 (red bars) instilled bladders revealed minimal MPO activity in controls, that is 11 ng/ml at 12 and 14 ng/ml at 24 hours, but significantly increased MPO activity in LL-37 instilled bladders with continued escalation from 229 ng/ml at 12 to 849 ng/ml at 24 hours.

Figure 4

SAGE GM-1111 treatment. Gross images of bladders precoated/treated with 10 mg/ml GM-1111, harvested at 24 hours and hemisected to expose inner mucosal surface before LL-37 instillation (A) and those with 10 mg/ml SAGE posttreatment after LL-37 instillation (B). Scale bar represents 0.5 μM (A and B). Tissue histological sections reveal PMNs (circles and ovals) and no significant evidence of PMN margination out of blood vessels (rectangle) in GM-111 pretreated (C) and SAGE posttreated (D) bladders. SMC, smooth muscle cell layer. LP, lamina propria. SBM, submucosa. Reduced from 10× (C and D). Scale bar represents 75 μM (C and D). Tissue MPO assay for GM-1111 precoating/treatment revealed 22-fold decrease in MPO activity in bladders instilled with heparin followed by LL-37 (purple bars) vs that in bladders instilled with LL-37 only (red bar) (p = 0.013) (E). Tissue MPO assay for SAGE posttreatment demonstrated 2.5-fold decrease in MPO activity in posttreated samples instilled with LL-37 only (purple bar) vs that in bladders instilled with LL037 followed by heparin (p = 0.220) (F).

Figure 5

Heparin treatment. Gross images of bladders harvested at 24 hours and hemisected to expose inner mucosal surface of bladders precoated/treated with 10 mg/ml heparin before LL-37 instillation (A) vs 10 mg/ml heparin posttreatment after LL-37 instillation (B). Scale bar represents 0.5 cm (A and B). Tissue histology shows bladders precoated/treated with 10 mg/ml heparin before LL-37 instillation (C) vs 10 mg/ml heparin posttreatment after LL-37 instillation (D). SMC, smooth muscle cell layer. LP, lamina propria. SBM, submucosa. Reduced from ×10 (C and D). Scale bar represents 75 μM (C and D). Tissue MPO assay revealed slightly decreased MPO activity in heparin precoated/treated bladders instilled with SAGE followed by LL-37 (purple bar) vs that in bladders instilled with LL-37 only (red bar) (E). Tissue MPO assay for heparin posttreatment showed no significant decrease in posttreated bladders instilled with LL-37 followed by SAGE (purple bar) vs that in bladders instilled with LL-37 only (red bar) (F). heparin posttreated tissues (LL-37, 849 vs 827 ng/ml, fig. 5, F). Grossly group 2 (pretreatment) showed similar findings (fig. 5, A). Histological findings mirrored those in group 1 (fig. 5, C). MPO assays revealed only slight differences in inflammatory activity between untreated LL-37 challenged bladders and group 2 heparin pretreated tissues (LL-37, 849 vs 759 ng/ml, fig. 5, E).

Figure 6

SAGE coating as bladder armor. Tissues immediately harvested tissues after 10 mg/ml GM-1111-Alexa Fluor-633 instillation show uniform coating (green fluorescent area) of urinary GAG layer adjacent to urothelium (U) along with deeper penetration into submucosa (SBM), lamina propria (LP) and superficial smooth muscle (SMC) (A and B). Endothelial cells lining arterioles demonstrated significant GM-1111-Alexa Fluor 633 coating on basal and luminal sides (rectangles). Reduced from ×10 (A) and ×20 (B). Scale bars represent 75 (A) and 37.5 (B) μM. Tissues harvested 24 hours after 10 mg/ml SAGE instillation show no evidence of GM-1111-Alexa Fluor 633 along urinary GAG layer but coating was still strongly apparent in select submucosal regions and along endothelium lining small arterioles on basal and luminal sides (rectangles) (C and D). Coating was still noted intercalating in random smooth muscle cell layer regions. Reduced from ×20 (C and D). Scale bar represents 75 μM (C and D).