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 at 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.

Amplifying renal immunity: the role of antimicrobial peptides in pyelonephritis

“…Following a challenge with 320 μM intravesical LL-37 (a concentration ~65 times greater than the minimal inhibitory concentration against UPEC), bladders from C57Bl/6 mice showed urothelial ulceration, oedema and increased inflammatory cell infiltrate. 65 In addition, lower concentrations of LL-37 (5-10 μM) were cytotoxic to the urothelium and induced urothelial apoptosis. 66 Thus, additional studies are needed to assess the cytotoxicity profile of LL-37, define its molecular and cellular mechanisms, and examine the epithelial and haematopoietic sources of its production during UTI.…”

Prevention of sinonasal inflammation by a synthetic glycosaminoglycan

“…[20,32] The number of mast cells infiltrating into the tissue were quantified using a staining protocol for tryptase (rabbit anti-mast cell tryptase (1:800 dilution, Mast Cell Tryptase Antibody, sc-32889, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.); biotinylated secondary antibody (1:2000 dilution, goat anti-rabbit IgG-B, sc-2040, Santa Cruz Biotechnology, Inc., Santa Cruz, CA); 3,3′-diaminobenzidine (DAB) peroxidase substrate). Five randomly selected high-power (400×) fields for each tissue were imaged, and the number of mast cells was quantified.…”

“…Five randomly selected high-power (400×) fields for each tissue were imaged, and the number of mast cells was quantified. [20,32,41] The final results are presented as mast cell numbers per mm 2 sample.…”

“…1) previously demonstrated to be effective in attenuating inflammatory responses in rosacea,[19] initial cystitis (IC),[20,21] and periodontitis. [22] We therefore hypothesized that GM-0111 could prevent sinonasal inflammation in a mouse model of rhinosinusitis (RS).…”

Reviewing Interstitial Cystitis Models and Treatments: A Focus on the Urothelium

“…Continuing, it was also found that the intravesical instillation of cathelicidin, an antimicrobial peptide, and LL-37, C-terminal peptide fragment of the human cathelicidin, induces Hunner's lesions in the mucosa, acute inflammation, marked edema in all layers of the bladder and infiltration of polymorphonuclear leukocytes in healthy animal models (28). Other researchers also demonstrated that moderate stress, induced by exposing mice to constant illumination for 96 hours, resulted in desquamation of superficial and intermediate urothelial cells (29).…”