Decreased Inflammatory Response of Urothelial and Smooth Muscle Cells to Lipopolysaccharides by P75NTR Antagonism

Desormeau B1, Yan R L1, Mossa A1, Cammisotto P2, Campeau L3

Research Type

Pure and Applied Science / Translational

Abstract Category

Pharmacology

Abstract 339
ePoster 5
Scientific Open Discussion Session 21
On-Demand
Cell Culture Infection, Urinary Tract Painful Bladder Syndrome/Interstitial Cystitis (IC) Molecular Biology Pharmacology
1. McGill University, 2. Lady Davis Institute for Medical Research, 3. Jewish General Hospital
Presenter
L

Lysanne Campeau

Links

Abstract

Hypothesis / aims of study
Current evidence links acute urinary tract infection by E. coli to the progression of interstitial cystitis. As it appears, lipopolysaccharides, found on gram negative bacteria activate TLR4 and induce proinflammatory response in cells. Notably, higher tissue contents of the pro-nerve growth factor (proNGF) have been detected in patients with inflammation and sepsis. ProNGF activates p75NTR receptor and promotes cell death and degeneration in different tissues. As both receptors are present in normal bladder, we sought to identify a signalling interplay between both receptors in bladder cells and hypothesized that p75NTR antagonism could counteract TLR4 inflammatory pathways upon engagement by LPS in bladder cells.
Study design, materials and methods
Primary urothelial (UTC) and smooth muscle cells (SMC) from female Sprague-Dawley rats were cultured in media of LPS (0.001-100 µg/ml) individually or combined to THX-B (4 µg/ml). The intracellular and extracellular domains of p75NTR were detected by immunostaining and Western blotting. Downstream activation of NF-kB and MAPKs inflammatory/survival pathways after docking of the TRAF6 protein adaptor on p75NTR was quantified by immunoprecipitation and immunoblotting. As an index of apoptosis and inflammation, caspases 3 and 8 and iNOS enzymatic activities were evaluated by colorimetric assays. The amounts of proNGF, TNF-α and IL-1β produced and secreted in cell culture media were measured using ELISA kits.
Results
Protein expression of the p75NTR domains was localised in UTC and SMC independently of increasing LPS concentrations. The amounts of proNGF released was increased by low concentrations of LPS (0.001-0.1 µg/ml), then decreased past 1 µg/ml LPS in both types of cell. Caspase 3 and 8 activities were not detected in UTC and SMC with LPS or THX-B. The quantified ratio of pERK/ERK was significantly higher in UTC when exposed to LPS alone or in combination with THX-B comparatively to control. LPS increased TNF-α and NO in UTC and the addition of THX-B reduced the amounts of TNF-α only. While this was not observed in SMC, an increase in TRAF6 activation, NF-kB translocation and JNK phosphorylation by LPS was nonetheless detected in these cells; however, only the ratio of pJNK/JNK was decreased by THX-B treatment.
Interpretation of results
Our results support that both TLR4 and p75NTR contribute to inflammation in cystitis. It is demonstrated that, in vitro, the production of proNGF is LPS concentration-dependently affected: low LPS concentrations prevent proNGF maturation, whereas higher concentrations reverse this. It is showed further that p75NTR antagonism holds the potential to counteract LPS-induced production/secretion of TNF-α in UTC, as well as activation of the JNK inflammatory pathway in SMC without interfering with cell survival.
Concluding message
As previous investigations report a correlation between bacterial cystitis and the progression of chronic interstitial cystitis, the present study highlights new molecular targets to prevent this process. Blocking p75NTR may have an anti-inflammatory effect in bladder cells, though this needs to be validated in vivo.
Figure 1
Figure 2
Disclosures
Funding Canadian Urological Association Clinical Trial No Subjects Animal Species Rat Ethics Committee Animal Ethics Committee of McGill University
19/01/2022 09:34:12