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PURPOSE: Totally implantable ports require regular maintenance to prevent port-related complications. Manufacturers recommend monthly maintenance port flushes for patients for the life of the port. Previous studies show that extending intervals between maintenance port flushes up to 16 weeks does not increase incidence of port-related complications. To date, no prospective study has been conducted to evaluate the medical safety of extending flush intervals from monthly to every 12 weeks within a heterogeneous disease cohort. Research Question: Is it feasible and medically safe to extend intervals between maintenance port flushes to every 12 weeks in patients with cancer not on active treatment? PATIENTS AND METHODS: This study enrolled oncology and hematology patients who had retained their port following completion of anticancer treatment. Clinical data were extracted for 1,059 participants. The primary end points of this study were the overall number of ports removed and incidence of port-related complications reported between cohorts 1 and 2 (flushes every 4-8 weeks), and cohort 3 (flushes every 12 weeks). RESULTS: Data were allocated into three study cohorts on the basis of year and duration between port flushes. No difference was observed in the overall percentage of ports removed because of physician-reported complications across all cohorts (25%-30%). No change in the incidence of port-related complications including suspected infection and malfunction was observed between cohorts 1 and 2 (8%), or cohort 3 (5%). CONCLUSION: Our findings show that extending maintenance port flush intervals to 12 weeks does not increase the incidence of port-related adverse events and is medically safe.

Joint Bone Spine - In Press.Proof corrected by the author Available online since samedi 25 aout 2018

© 2017 Elsevier Ltd Autophagy is a ubiquitous cellular mechanism for the targeted lysosomal degradation of various cytosolic constituents, from proteins to organelles. As an essential homeostatic mechanism, autophagy is upregulated in response to numerous environmental and pharmacological stimuli, including starvation, where it facilitates the recycling of essential amino acids. In addition, autophagy plays specific roles within the immune system; it serves as a source of peptides for antigen presentation, a mechanism for the engulfment and degradation of intracellular pathogens and as a key regulator of inflammatory cytokines. In particular, autophagy has been shown to play a number of roles in regulating inflammasome activation, from the removal of inflammasome-activating endogenous signals, to the sequestration and degradation of inflammasome components. Autophagy also plays a role in determining the fate of IL-1β, which is concentrated in autophagosomes. This review discusses a growing body of literature that suggests autophagy is a critical regulator of inflammasome activation and the subsequent release of IL-1 family cytokines.

MIF is a key regulator of host immune responses and increased levels secreted from cells, or found circulating systemically, have been implicated in the pathogenesis of many inflammatory and autoimmune disorders. Here, we describe methods for detecting and quantifying extracellular concentrations of MIF in both human- and murine-derived biological samples.

Macrophage migration inhibitory factor (MIF) is expressed and released ubiquitously by numerous cell types and tissues. MIF is detected and constitutively expressed at the protein level both intra- and extracellularly. This chapter outlines methods for cultivating, purifying, detecting, and quantifying concentrations of MIF from murine primary derived macrophages and dendritic cell culture supernatants.

MIF is a key regulator of host immune responses and increased levels secreted from cells, or found circulating systemically, have been implicated in the pathogenesis of many inflammatory and autoimmune disorders. Here, we describe methods for detecting and quantifying extracellular concentrations of MIF in both human- and murine-derived biological samples.

Macrophage migration inhibitory factor (MIF) is expressed and released ubiquitously by numerous cell types and tissues. MIF is detected and constitutively expressed at the protein level both intra- and extracellularly. This chapter outlines methods for cultivating, purifying, detecting, and quantifying concentrations of MIF from murine primary derived macrophages and dendritic cell culture supernatants.

Macrophages are professional phagocytes that display remarkable plasticity, with a range of phenotypes that can be broadly characterized by the M1/M2 dichotomy. Glucocorticoid (GC)-induced leucine zipper (GILZ) is a protein known to mediate anti-inflammatory and some pro-resolving actions, including as neutrophil apoptosis. However, the role of GILZ in key macrophage function is not well understood. Here, we investigated the role of GILZ on macrophage reprogramming and efferocytosis. Using murine bone-marrow-derived macrophages (BMDMs), we found that GILZ was expressed in naive BMDMs and exhibited increased expression in M2-like macrophages (IL4-differentiated). M1-like macrophages (IFN/LPS-differentiated) from GILZ-/- mice showed higher expression of the M1 markers CD86, MHC class II, iNOS, IL-6 and TNF-α, associated with increased levels of phosphorylated STAT1 and lower IL-10 levels, compared to M1-differentiated cells from WT mice. There were no changes in the M2 markers CD206 and arginase-1 in macrophages from GILZ-/- mice differentiated with IL-4, compared to cells from WT animals. Treatment of M1-like macrophages with TAT-GILZ, a cell-permeable GILZ fusion protein, decreased the levels of CD86 and MHC class II in M1-like macrophages without modifying CD206 levels in M2-like macrophages. In line with the in vitro data, increased numbers of M1-like macrophages were found into the pleural cavity of GILZ-/- mice after LPS-injection, compared to WT mice. Moreover, efferocytosis was defective in the context of GILZ deficiency, both in vitro and in vivo. Conversely, treatment of LPS-injected mice with TAT-GILZ promoted inflammation resolution, associated with lower numbers of M1-like macrophages and increased efferocytosis. Collectively, these data indicate that GILZ is a regulator of important macrophage functions, contributing to macrophage reprogramming and efferocytosis, both key steps for the resolution of inflammation.

Background Systemic sclerosis (SSc) is an autoimmune connective tissue disorder, the pathogenesis of which remains unknown. Recent evidence suggests dysregulation of the innate immune system, particularly interleukin-(IL)−1 family cytokines. Given the emerging role of macrophage migration inhibitory factor (MIF) in pathways of IL-1 family cytokine secretion, the role of MIF, IL-1α, IL-1β and IL-18 in SSc is of interest. Objectives To examine associations between MIF and IL-1 cytokines (IL-1α, IL-1β, IL-18), in SSc, and associations with clinical features. Methods 115 SSc patients (2013 ACR/EULAR criteria) attending Monash Scleroderma Clinic and 52 healthy controls were recruited between August 2015 and August 2017. Serum MIF, IL-1α, IL-1β and IL-18 levels were quantified using ELISA and analysed alongside concurrent clinical and laboratory data from the Australian Scleroderma Cohort Study database Results Compared to controls, SSc patients had significantly elevated serum MIF and IL-18 (figure 1). A weak positive correlation was observed between MIF and SScHAQ score (R=0.2107, p=0.0437) which was stronger in the diffuse cutaneous (dcSSc) subgroup (R=0.457, p=0.0373). Patients with elevated IL-18 levels were more likely to have active disease (EUSTAR score ≥3) however IL-18 was lower in patients with pulp atrophy and sclerodactyly. IL-1β was elevated in dcSSc patients with pulmonary fibrosis and correlated with mRSS (R=0.213, p=0.0254) in all SSc patients. IL-1α was elevated in patients with joint contractures and pulp atrophy. Positive correlations were found between concentrations of MIF and both IL-1α and IL-1β. However, there was no significant correlation between MIF and IL-18. Conclusions MIF and IL-18 were significantly elevated in SSc compared to health controls, and IL-1 family cytokines were variably associated with clinical manifestations of SSc. A relationship between MIF and IL-1β was confirmed. Further investigation into the roles of MIF and IL-1 family cytokines in SSc is justified. References [1] Zhang L, Yan JW, Wang YJ, Wan YN, Wang BX, Tao JH, et al. Association of interleukin 1 family with systemic sclerosis. Inflammation2014;37(4):1213–20. [2] van Bon L, Cossu M, Radstake TR. An update on an immune system that goes awry in systemic sclerosis. Curr Opin Rheumatol2011;23(6):505–10. Disclosure of Interest None declared

Background and Aims Macrophage migration inhibitory factor (MIF) is a mediator of innate immunity and is implicated in the pathogenesis of numerous autoinflammatory disorders. Clinical studies have shown correlations between elevated levels of IL-1β and MIF in serum with disease outcomes of patients with autoimmune disorders. To date, it is unclear whether MIF specifically regulates the expression and secretion of IL-1 family cytokines. Therefore, we aim to characterise mechanisms by which MIF may modulate the secretion of IL-1 family cytokines. Methods The biological activity of MIF in murine bone marrow derived macrophages (BMDM) was inhibited using a small molecule inhibitor, COR123625. Concurrently, BMDM derived from Mif-/- mice were employed to evaluate the effects of MIF depletion on regulation of IL-1 family cytokines. mRNA expression was analysed by qRTPCR. Protein expression and secretion of IL-1α, IL-1β and IL-18 was assessed by western blot and ELISA. Results We show that depletion of MIF in macrophages significantly reduced IL-1 cytokine release specifically in response to NLRP3 stimuli, but has no effect on the secretion of TNF-α and IL-6. Moreover, diminished IL-1 responses were independent of production of pro-IL-1β. Instead, MIF depletion specifically inhibits NLRP3-mediated IL-1 responses as levels were unaffected following activation of AIM2 or NLRC4 inflammasomes. Conclusions Our findings reveal a novel role for MIF in the modulation of IL-1-dependent inflammatory responses, linking MIF directly to NLRP3 inflammasome activation. This study for the first time implicates a specific role for MIF in the release of IL-1 family cytokines and highlights the potential of targeting MIF in IL-1-dependent pathologies.

Infectious epididymitis in men, a frequent entity in urological outpatient settings, is commonly caused by bacteria originating from the anal region ascending the genitourinary tract. One of the most prevalent pathogens associated with epididymitis is Escherichia coli . In our previous study, we showed that semen quality is compromised in men following epididymitis associated with specific E. coli pathovars. Thus, our aim was to investigate possible differences in immune responses elicited during epididymitis following infection with the uropathogenic E. coli (UPEC) strain CFT073 and the nonpathogenic enteric E. coli (NPEC) strain 470. Employing an in vivo experimental epididymitis model, C57BL/6 mice were infected with UPEC CFT073, NPEC 470, or phosphate-buffered saline (PBS) as a sham control for up to 7 days. After infection with NPEC 470, the expression of proinflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha in the epididymis was significantly increased. Conversely, UPEC CFT073-challenged mice displayed inflammatory gene expression at levels comparable to sham PBS-treated animals. Moreover, by day 7 only NPEC-infected animals showed activation of adaptive immunity evident by a substantial influx of CD3 + and F4/80 + cells in the epididymal interstitium. This correlated with enhanced production of Th1-associated cytokines IL-2 and gamma interferon (IFN-γ). Furthermore, splenocytes isolated from UPEC-infected mice exhibited diminished T-cell responses with significantly reduced secretion of IL-2 and IFN-γ in contrast to NPEC-infected animals. Overall, these findings provide new insights into understanding pathogen-specific modulation of host immunity during acute phases of epididymitis, which may influence severity of disease and clinical outcomes.

Autophagy is a highly conserved homeostatic mechanism for the lysosomal degradation of cytosolic constituents, including proteins and organelles. Studies over the past 10 years have demonstrated a number of specific roles for autophagy in the regulation of host immune responses, host–microbe interactions, and inflammation. This chapter presents some of the known and proposed mechanisms through which autophagy impacts on inflammatory responses elicited by antigen-presenting cells, with a particular emphasis on the modulation of cytokine expression, production, and release. Through regulation of the IL-1 family cytokines, IL-1α, IL-1β, and IL-18, autophagy has far-reaching impacts on numerous immune cell types, in particular by influencing the secretion of IL-23 and IL-17. Autophagy intersects with IL-1β both directly, through sequestration of the cytokine in autophagosomes, and indirectly, through regulation of inflammasome activation in response to inflammatory stimuli. Perturbations in autophagic activity can also affect the secretion of other cytokines, including macrophage migration inhibitory factor. A better understanding of how autophagy exerts its effects on inflammation has the potential to open new therapeutic avenues for the treatment of diseases with inflammatory pathologies.

MIF (macrophage migration inhibitory factor [glycosylation-inhibiting factor]) is a pro-inflammatory cytokine expressed in multiple cells types, including macrophages. MIF plays a pathogenic role in a number of inflammatory diseases and has been linked to tumor progression in some cancers. Previous work has demonstrated that loss of autophagy in macrophages enhances secretion of IL1 family cytokines. Here, we demonstrate that loss of autophagy, by pharmacological inhibition or siRNA silencing of Atg5, enhances MIF secretion by monocytes and macrophages. We further demonstrate that this is dependent on mitochondrial reactive oxygen species (ROS). Induction of autophagy with MTOR inhibitors had no effect on MIF secretion, but amino acid starvation increased secretion. This was unaffected by Atg5 siRNA but was again dependent on mitochondrial ROS. Our data demonstrate that autophagic regulation of mitochondrial ROS plays a pivotal role in the regulation of inflammatory cytokine secretion in macrophages, with potential implications for the pathogenesis of inflammatory diseases and cancers.

Objectives Systemic sclerosis (SSc) is an autoimmune disease characterised by fibrosis, vascular dysfunction and immune dysregulation. The pathogenesis of SSc remains poorly understood, although studies have indicated a role for the innate immune response. Methods Here, we measured serum interleukin (IL)‐1α, IL‐1β and IL‐18 levels in 105 SSc patients and 47 healthy controls (HC) and analysed them with respect to multiple clinical parameters. Results Serum IL‐18 concentrations were significantly higher in SSc patients than in HC, while no significant differences in concentrations of IL‐1α and IL‐1β were observed between SSc and HC. In both SSc and HC serum, IL‐1α and IL‐1β were positively correlated, while in SSc, both cytokines negatively correlated with IL‐18. Serum IL‐18 was significantly negatively correlated with both carbon monoxide transfer coefficient (KCO) and diffusing capacity of the lungs for carbon monoxide (DLCO). Serum IL‐1β was positively correlated with the modified Rodnan skin score (mRSS), particularly in patients with limited subtype. DLCO, KCO and tricuspid regurgitation (TR) velocity were significantly higher in patients with high serum IL‐1β. Serum IL‐1α was significantly lower in SSc patients with low KCO and positively correlated with KCO. SSc patients with high serum IL‐1α concentrations were more likely to have digital ulcers. Conclusions Our data suggest that these IL‐1 family cytokines may have different roles in the pathogenesis of SSc fibrotic complications.

Toll-like receptors (TLRs) are essential mediators of both innate and adaptive immunity by recognizing and eliciting responses upon invasion of pathogens. The response of TLRs must be stringently regulated as exaggerated expression of signalling components as well as pro-inflammatory cytokines can have devastating effects on the host, resulting in chronic inflammatory diseases, autoimmune disorders and aid in the pathogenesis of TLR-associated human diseases. Therefore, it is essential that negative regulators act at multiple levels within TLR signalling cascades, as well as through eliciting negative-feedback mechanisms in order to synchronize the positive activation and negative regulation of signal transduction to avert potentially harmful immunological consequences. This review explores the various mechanisms employed by negative regulators to ensure the appropriate modulation of both immune and inflammatory responses.

Macrophage migration Inhibitory factor (MIF) was one of the earliest pro-inflammatory cytokines to be identified. Increasing interest in this cytokine in recent decades has followed the cloning of human MIF and the generation of Mif(-/-) mice. Deepening understanding of signaling pathways utilized by MIF and putative receptor mechanisms have followed. MIF is distinct from all other cytokines by virtue of its unique induction by and counter regulation of glucocorticoids (GCs). MIF is further differentiated from other cytokines by its structural homology to specific tautomerase and isomerase enzymes and correlative in vitro enzymatic functions. The role of MIF in immune and inflammatory states, including a range of human autoimmune diseases, is now well established, as are the relationships between MIF polymorphisms and a number of inflammatory diseases. Here, we review the known pleiotropic activities of MIF, in addition to novel functions of MIF in processes including autophagy and autophagic cell death. In addition, recent developments in the understanding of the role of MIF in systemic lupus erythematosus (SLE) are reviewed. Finally, we discuss the potential application of anti-MIF strategies to treat human diseases such as SLE, which will require a comprehensive understanding of the unique and complex activities of this ubiquitously expressed cytokine.

Patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) have suppressed TLR2 expression, function and cytokine production. The aim of this study was to explore the importance of hepatitis B virus (HBV) genotype in innate immune responses and investigate whether Toll-like receptor (TLR) expression/function has potential roles as predictive biomarkers of successful therapy with pegylated interferon (Peg-IFN) therapy of HBeAg seroconversion in HBeAg-positive patients. We showed that as early as 4 weeks after initiation of Peg-IFN, future HBeAg seroconverters had significantly elevated levels of TLR2 expression on monocytes. TLR2-associated IL-6 production at baseline and week 4 of therapy and TLR4 IL-6 production at week 4 were also markedly elevated in HBeAg seroconverters. HBV genotype also influenced treatment response, with genotypes A and B more likely to seroconvert than D. We were able to demonstrate that these differences were due in part to the interaction of the specific HBeAg proteins with TLR pathway adaptor molecules, and these interactions were genotype dependent. HBeAg-mediated modulation of TLR signalling was also observed in Huh7 cells, following stimulation with Pam3Cys. Importantly, the addition of IFN-α to TLR2-stimulated cells cotransfected with an HBeAg expression plasmid reversed HBeAg-mediated suppression of hepatocytes. These findings demonstrate that patients with an activated inflammatory response are much more likely to respond to IFN therapy, with TLR responses showing promise as potential biomarkers of HBeAg seroconversion in this setting. Furthermore, our findings suggest there is differential genotype-specific HBeAg suppression of innate signalling pathways which may account for some of the clinical differences observed across the CHB spectrum.

Objectives Macrophage migration inhibitory factor (MIF) and D‐dopachrome tautomerase (DDT), members of the same cytokine superfamily, are linked to the pathogenesis of a number of inflammatory diseases. The aim of this study was to investigate their clinical relevance in systemic sclerosis (SSc). Methods Serum MIF and DDT were quantified in 105 SSc patients by ELISA and levels compared to healthy controls (HC) (47) and patients with systemic lupus erythematosus (SLE) (184). Clinical parameters included organ involvement, serum laboratory markers and results of pulmonary function tests, and overall disease activity assessed using the European Scleroderma Trials and Research group (EUSTAR) activity index. Results There was no significant difference in serum DDT concentrations between patients with SSc and HC. However, serum MIF was significantly increased in SSc compared to both HC and SLE cohorts. Serum MIF was increased in SSc patients with low forced vital capacity (FVC) and was also associated with the use of angiotensin II receptor blockers and beta blockers in SSc, confirmed after adjusting for the presence of systemic hypertension and low FVC. Serum DDT was significantly higher in SSc patients with low FEV1 and negatively correlated with EUSTAR score, particularly in patients with limited disease. Conclusion Although not significantly linked to specific clinical parameters, serum MIF was significantly higher in SSc patients than in HC and SLE patients, suggesting a fundamental role for MIF in SSc. DDT, while closely related to MIF, did not show a similar expression profile, suggesting functional differences between these molecules.

Uropathogenic Escherichia coli (UPEC)-associated epididymitis is commonly diagnosed in outpatient settings. Although the infection can be successfully cleared using antimicrobial medications, 40% of patients unexplainably show persistent impaired semen parameters even after treatment. Our aim was to investigate whether pathogenic UPEC and its associated virulence factor hemolysin (hlyA) perturb the structural and functional integrity of both the epididymis and sperm, actions that may be responsible for the observed impairment and possibly a reduction of fertilization capabilities. Semen collected from patients diagnosed with E. coli-only related epididymitis showed that sperm counts were low 14 days postantimicrobial treatment regardless of hlyA status. At Day 84 following treatment, hlyA production correlated with approximately 4-fold lower sperm concentrations than in men with hlyA-negative strains. In vivo experiments with the hlyA-producing UPEC CFT073 strain in a murine epididymitis model showed that just 3 days postinfection, structural damage to the epididymis (epithelial damage, leukocyte infiltration, and edema formation) was present. This was more severe in UPEC CFT073 compared to nonpathogenic E. coli (NPEC 470) infection. Moreover, pathogenic UPEC strains prematurely activated the acrosome in vivo and in vitro. Raman microspectroscopy revealed that UPEC CFT073 undermined sperm integrity by inducing nuclear DNA damage. Consistent with these observations, the in vitro fertilization capability of hlyA-treated mouse sperm was completely abolished, although sperm were motile. These findings provide new insights into understanding the possible processes underlying clinical manifestations of acute epididymitis.

Uropathogenic Escherichia coli (UPEC)-associated epididymitis is commonly diagnosed in outpatient settings. Although the infection can be successfully cleared using antimicrobial medications, 40% of patients unexplainably show persistent impaired semen parameters even after treatment. Our aim was to investigate whether pathogenic UPEC and its associated virulence factor hemolysin (hlyA) perturb the structural and functional integrity of both the epididymis and sperm, actions that may be responsible for the observed impairment and possibly a reduction of fertilization capabilities. Semen collected from patients diagnosed with E. coli-only related epididymitis showed that sperm counts were low 14 days postantimicrobial treatment regardless of hlyA status. At Day 84 following treatment, hlyA production correlated with approximately 4-fold lower sperm concentrations than in men with hlyA-negative strains. In vivo experiments with the hlyA-producing UPEC CFT073 strain in a murine epididymitis model showed that just 3 days postinfection, structural damage to the epididymis (epithelial damage, leukocyte infiltration, and edema formation) was present. This was more severe in UPEC CFT073 compared to nonpathogenic E. coli (NPEC 470) infection. Moreover, pathogenic UPEC strains prematurely activated the acrosome in vivo and in vitro. Raman microspectroscopy revealed that UPEC CFT073 undermined sperm integrity by inducing nuclear DNA damage. Consistent with these observations, the in vitro fertilization capability of hlyA-treated mouse sperm was completely abolished, although sperm were motile. These findings provide new insights into understanding the possible processes underlying clinical manifestations of acute epididymitis.

Background & Aims Viruses target innate immune pathways to evade host antiviral responses. Recent studies demonstrate a relationship between hepatitis B disease states and the host's innate immune response, although the mechanism of immunomodulation is unknown. In humans, the innate immune system recognizes pathogens via pattern recognition receptors such as the Toll-like receptors (TLR), initiating anti-inflammatory responses. TLR expression and pro-inflammatory cytokine production is reduced in hepatitis B e antigen (HBeAg)-positive patients following TLR stimulation. The aim of this study was to investigate interactions between TLR signaling pathways and the mature HBeAg protein localized in the cytosol. Methods The ability of HBeAg to inhibit TLR signaling and association with TLR adapters was evaluated by immunoprecipitation, immunostaining, and reporter studies. Results Our findings show that HBeAg co-localizes with Toll/IL-1 receptor (TIR)-containing proteins TRAM, Mal, and TLR2 at the sub-cellular level, which was not observed for Hepatitis B core antigen. Co-immunoprecipitation analysis demonstrated HBeAg interacted with TIR proteins Mal and TRAM, while a mutated HBeAg ablated interaction between Mal and MyD88. Importantly, HBeAg also disrupted homotypic TIR:TIR interaction critical for TLR-mediated signaling. Finally, HBeAg suppressed TIR-mediated activation of the inflammatory transcription factors, NF-κB and Interferon-β promoter activity. Conclusions Our study provides the first molecular mechanism describing HBeAg immunomodulation of innate immune signal transduction pathways via interaction and targeting of TLR-mediated signaling pathways. These finding suggest the mechanism as to how HBeAg evades innate immune responses contributing to the pathogenesis of chronic hepatitis B infection and the establishment of viral persistence.

Introduction 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- κ B 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. Methods To evaluate the consequences of hlyA on cytokine production in vitro, RAW 267.4 cells or single epididymides dissected from C57BL/6 N mice were left untreated, treated with LPS (100 ng/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/6 N mice. PBS injections were used as a sham control for in vivo experiments. Following 3 days 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. Results 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 was comparable to PBS infected mice, whereas NPEC 470 significantly increased IFN-? expression. In addition, a decrease in the number of F4/80 and CD3+ cells within the epididymal interstitium of UPEC CFT073 infected mice was observed in comparison to NPEC 470 infected mice. Conclusion Overall these finding suggest that hlyA produced by UPEC attenuates host innate immune responses in the initial phases of infection for successful colonisation within the epididymis. Furthermore, the down-regulation of IFN-? attenuates maturation of adaptive immune responses thereby evading elimination and contributing to persistence.