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.
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.