P032 Alpha-haemolysin differentially modulates innate immune responses in the experimental murine epididymitis model

Infection and inflammation of the urinary tract are aetiological factors contributing to 10–15% of infertility cases in men. Epididymitis is often the consequence of ascending bacterial infection into the urinary tract. Specific strains of Uropathogenic E. coli (UPEC) produce the toxin alpha-haemolysin (hlyA), which has been shown to manipulate host immune responses by degrading components of the NF-κB signalling cascade following infection of bladder epithelial cells. The aim of this study was to elucidate the consequences of hlyA production on innate immune responses in the UPEC induced murine experimental epididymitis model. To evaluate the consequences of hlyA on cytokine production in vitro, RAW 267.4 cells or single epididymides dissected from C57BL/6N mice were left untreated, treated with LPS (100ng/mL) or infected with the clinically relevant pyelonephritic?-haemolytic (UPEC CFT073) or non-haemolytic E. coli strain NPEC 470. Cell supernatants were collected following treatment and cytokine production analysed using the BD™Cytometric Bead Array. The establishment of the murine experimental epididymitis model was achieved through injection of 40,000 UPEC CFT073 or NPEC 470 in total, into both left and right vas deferens of C57BL/6N mice. PBS injections were used as a sham control for in vivo experiments. Following 3days infection, epididymides were dissected and snap frozen. qPCR analyses was performed to quantify pro-inflammatory gene expression. Immuno-fluorescent staining was used for the detection of macrophages (F4/80) and T-cells (CD3). Statistical analysis was performed by One-way ANOVA followed by the Bonferroni’s Multiple Comparison Test where appropriate. P⩽0.05 is considered statistically significant. In this study we show that the?-haemolytic UPEC strain CFT073 significantly reduced secretion of pro-inflammatory cytokines TNF-?, IL-6, IL-1 and IFN-? in epididymal organ cultures and RAW 267.4 cells following infection. Conversely, in vivo qPCR analyses revealed expression of early response genes TNF-? and IL-6 were significantly increased by day 3 following UPEC CFT073 infection. Interestingly, RANTES/CCL5 and IFN-? gene expression were differentially modulated dependent on the hylA status of the E. coli strain. Our findings illustrate that hlyA production significantly enhances RANTES expression following UPEC CFT073 infection in contrast to PBS and NPEC 470 infected mice. Conversely, IFN-? gene expression in UPEC CFT073 infected mice wa