Investigations of the innate immune defences in the urogenital tract

Abstract

The urinary tract (UT) is a sterile environment despite being constantly challenged by pathogens from its surrounding environment. Protection of the uroepithelium from microbial assault involves the innate immune system, but still, to date, little is known of the actual mechanisms that function in such defences. The focus of the research presented in this thesis, which used RT4 and VK-2 E6/E7 in vitro cell systems to model the bladder and vaginal epithelia respectively, was to interrogate the mechanisms by which these epithelia recognise and respond to microbial assault. The RNA of bladder RT4 cells challenged with motile and non-motile Escherichia coli strains was analysed via a TLR related gene array. The data supported upregulation of the NF-κB dependent cytokine IL-8 and thus NF-κB signalling was used to measure the uroepithelial response to microbial infection. Flagellin, recognised by TLR-5, and responsible for bacterial motility, induced a significant (p<0.001), 44.9±1.7 fold increase, in RT4 NF-κB signalling within 4h of challenge and with use of TLR-5 blocking antibody was identified as the key activating PAMP in the UT. Other bacterial cell wall components, namely LPS and peptidoglycan, failed to induce comparable NF-κB activation (maximum 10.7± 0.8 and 6.3±0.6 fold increase at 16 hours post challenge). These data led to the hypothesis that strains associated with urinary tract infection (UTI) have reduced or no motility. However, 12/24 clinical strains associated with UTI were motile and induced a NF-κB signalling response. Variability in the responses (3.4 0.3 to 40.3 1.8 fold at 8 hours post challenge), could not be explained by flagellin, suggesting that other factors, host and bacterial, may also contribute to the intensity of the response. Analysing UPEC strains isolated from the urine of patients carrying a C1174T SNP encoding a truncated TLR-5 revealed such strains to induce normal NF-κB signalling (39.5 0.3 fold) in RT4 cells. These data emphasised the importance of TLR-5 and bacterial motility in the innate response of the host to a UTI. Human (h) βD-2, a member of the Defensin family with microbial killing properties, is important in the innate defences of the uroepithelium. To identify agents that could be used therapeutically to enhance the innate defences of the UT, a reporter construct (phβD-2-Luc), containing 2 kbp of hβD- 2 5’UTR sequence, was transfected into RT4 and VK-2 E6/E7 vaginal cells. Reporter activity (fold increase) was significantly increased with calcitriol (VitD, 10 nM), or 17β-oestradiol (17β-oes, 4 nM), treatments when challenging VK-2 E6/E7 with either flagellin (68.1±8.5 vs. 196.3±28.3 for VitD, p<0.001, or vs. 199.5 37.7 for 17β-oes, p<0.01) or E. coli NCTC 10418(19.9±3.8 vs. 40.1±4.3 for VitD, p<0.01) or vs. 40.2±6.6 for 17β-oes, p<0.05). The hβD-2 reporter data also revealed Zymosan (50 μg/ml) enhancing reporter activity in both cell lines (RT4 34.8±5.0 and VK-2 E6/E7 27.2±2.9, both p<0.001) and had a synergistic effect to flagellin increasing reporter activity by more than 60 % (p<0.05). These data suggested that Zymosan, vitamin D as well as oestrogen regulate the hβD-2 gene. The expression of hβD-3, another member of the Defensin family with microbial killing properties, was identified in human bladder biopsies so its expression was further investigated in vitro. While no hβD-3 expression was measured in the RT4 cells, constitutive and inducible expression was identified in VK-2 E6/E7 cells. Human βD-3 expression was enhanced at 8 hours following challenge of the cells (PBS 70.5±13.4) with E. coli NCTC 10418 (1094.5±293.8, p<0.001), flagellin (518±139.2, p<0.01) and Zymosan (280.8±65.7, p<0.01). Induction was also observed with LPS but at 16 hours (27.0±8.1 (PBS), 612.2±260.8, p<0.001) but not with peptidoglycan (32.2±12.3, p>0.05). These data suggested that hβD-3 functions in the defence of urogenital tract and as its expression was inducible, a potential target for therapeutic strategies. The fungal cell wall β1,3-glucan, Zymosan, induced NF-κB signalling as well as hβD-2 in the RT4 and VK-2 E6/E7 cells, and hβD-3 expression and secretion in VK-2 E6/E7 cells. Molecular analysis of the cell RNA identified the expression of three isoforms (417, 330, 211 bp) of the Dectin-1 (CLEC7A) receptor and immunocytochemistry identified the receptor protein localised to the cell membrane. Following Zymosan challenge of VK-2 E6/E7 cells, receptor clustering and the phosphorylation of a Dectin-1 signalling protein, SYK was observed supporting the presence of functional receptors. Induction of NF-κB signalling by Zymosan was inhibited by blocking TLR-5 (4.2±0.4 vs 2.8±0.1, p<0.01) suggesting functional interactions between Dectin-1 and TLR-5. These data provide support for the presence of Dectin-1 receptor in the urogenital tract. In conclusion, the data presented in this thesis confirms the importance of TLR-5 in the defence of the urogenital tissues. Investigations of hβD-2 and hβD-3 expression and their enhancement in the UT have led to the discovery of a role for the Dectin-1 receptor. This receptor represents a target for future therapeutic strategies to enhance the innate response