Wanjian Jia

A Murine Model of Inflammatory Bladder Disease: Cathelicidin Peptide Induced Bladder Inflammation and Treatment With Sulfated Polysaccharides

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.

Prevention of Anti-microbial Peptide LL-37-Induced Apoptosis and ATP Release in the Urinary Bladder by a Modified Glycosaminoglycan

Interstitial cystitis (IC), often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG) layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1) the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2) the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3) the physiological and molecular markers that correlate with the severity of the inflammation, and (4) the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.

Topical cathelicidin (LL-37) an innate immune peptide induces acute olfactory epithelium inflammation in a mouse model.

Cathelicidin (LL‐37) is an endogenous innate immune peptide that is elevated in patients with chronic rhinosinusitis (CRS). The role of LL‐37 in olfactory epithelium (OE) inflammation remains unknown. We hypothesized that: (1) LL‐37 topically delivered would elicit profound OE inflammation; and (2) LL‐37 induced inflammation is associated with increased infiltration of neutrophils and mast cells.

IL-33 mast cell axis is central in LL-37 induced bladder inflammation and pain in a murine interstitial cystitis model

Graphical abstract Figure. No Caption available. HighlightsMast cells play a key role in IC/PBS & in our murine model of LL‐37 induced cystitis.IL‐33 appears to be a key cytokine in mast cell activation within the bladder.Mast cell deficient mice demonstrate diminished bladder inflammation and pain.IL‐33 is upregulated in the bladder after LL‐37 exposure.Targeting the IL‐33 – mast cell axis may be promising for future therapeutics. &NA; Interstitial cystitis (IC), also known as painful bladder syndrome (PBS), is a debilitating chronic condition that afflicts over 3 million women above the age of 18 in the U.S., and most patients fail to respond to current treatment options. Mast cells have previously been implicated as both a diagnostic and prognostic marker in IC/PBS. Patients with IC/PBS have been shown to have elevated levels of IL‐33, a cytokine released in response to tissue insult, in their urine. We hypothesize that mast cell‐mediated inflammation induced from IL‐33 may play an important role in initiating pain and inflammation in IC/PBS. A human cathelicidin, LL‐37, which is found at elevated levels in IC/PBS patients, was used to induce an IC/PBS‐like state of inflammation and bladder pain in mast cell deficient C‐kit (−/−) and wild type C57Bl/6 (WT) mice. Inflammation was quantified using myeloperoxidase (MPO) expression in bladder tissues measured via ELISA. Response rate to suprapubic stimulation from von Frey filaments was used to assess the relative pain and discomfort. Both types of mice increased IL‐33 expression in response to LL‐37 exposure. However, mast cell deficient mice demonstrated significantly lower levels of inflammation (p < 0.001) and reduced pain response (p < 0.001) compared to WT mice. These findings implicate an IL‐33‐mast cell dependent axis with a potential etiology of pain and inflammation in IC/PBS. Future therapeutics aimed at targeting the IL‐33 ‐ mast cell axis could potentially serve as useful targets for treating IC/PBS.

MP09-05 SILK-ELASTINLIKE POLYMERS ENHANCE THE ANTI-INFLAMMATORY AND ANALGESIC PROPERTIES OF SEMISYNTHETIC GLYCOSAMINOGLYCANS

Semisynthetic Glycosaminoglycans M. Martin Jensen1,2, Wanjian Jia2,3, Austin J. Schults3, Kyle J. Isaacson1,2, Douglas Steinhauff1,2, Bryant Green1,2, Marcelo Correa1,2, Jeremiah A. Alt4, Joseph Cappello4, Hamid Ghandehari1,2,5, Siam Oottamasathien2,3,6,7 1Department of Bioengineering, University of Utah 2Utah Center for Nanomedicine, Nano Institute of Utah 3Department of Medicinal Chemistry, University of Utah 4Department of Surgery, University of Utah School of Medicine 5Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah 6Department of Surgery and Division of Pediatric Urology, Primary Children’s Medical Center, Salt Lake City, Utah, USA 7Department of Pediatric Surgery and Division of Pediatric Urology, Massachusetts General Hospital for Children/Harvard Medical School, Boston, MA, USA