Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coliimplications for urinary tract infection. During the course of urinary tract infection (UTI) nitric oxide (NO) is generated as part of host response. The aim of this study was to investigate the significance of the NO-detoxifying enzymes flavohemoglobin (Hmp) and flavorubredoxin (Norv) in protection of uropathogenic Escherichia coli (UPEC) against nitrosative stress. Hmp (J96∆hmp) and norV (J96∆norV) knockout mutants of UPEC strain J96 were constructed using a single-gene deletion strategy. Bacterial tolerance and expression of hmp and norV in response to the NO-donor DETA/NO was evaluated in Luria broth and urine from healthy volunteers. Bacterial NO consumption and respiratory inhibition were assessed when exposed to NO. Expression of hmp and norV from E. coli originating from patients with UTI was evaluated using real-time PCR. The colonizing ability of J96 wild-type (wt) compared to an hmp-deficient mutant was assessed using a competition-based mouse UTI model. The viability of J96∆hmp and J96∆norV was significantly reduced compared to the wildtype strain after exposure to DETA/NO. The hmp expression in DETA/NO-exposed cultures was similar in J96wt and J96∆norV, while J96∆hmp showed an increased norV expression compared to J96wt. The NO consumption in J96∆hmp, but not in J96∆norV, was significantly impaired compared to J96wt. An up-regulation of hmp expression was found in E. coli isolated from all UTIpatients while norV expression increased in 50% of the patients. In the mouse UTI model, the hmp-mutant strain was significantly out-competed by the wild-type strain in the bladder and kidney. Hmp and NorV contribute to the protection of UPEC against NOmediated toxicity in vitro. Screening UPEC isolates from UTI patients revealed an increased hmp expression in all patients which confirms that hmp expression occurs in vivo in the infected human urinary tract. The ability to colonize the mouse urinary tract was impaired in the hmp-deficient mutant compared to the wild-type strain. NO-detoxification by Hmp is suggested to be an important characteristic for UPEC in protection against nitrosative stress and may be a virulence-facilitating factor.

Extracellular ATP is released from a variety of cells not only as a consequence of cell injury or cell death but also via nonlytic mechanisms (25). ATP has attracted increasing attention as a danger signal released during the initial phase of infection and inflammation in order to trigger innate immunity (18). In a healthy bladder, release of ATP from uroepithelial cells by stretch and distension mediates the sensation of bladder fullness by activation of P2X 3 receptors on suburothelial sensory nerves (5). Pathophysiological conditions cause enhanced bladder release of ATP as shown in patients diagnosed with interstitial cystitis (29,30,31). Although several studies have shown that pathology often results in augmented ATP release from the urothelium, there have not been any studies of ATP release from urothelium infected with bacteria. Patients with bacteriuria have increased urinary levels of ATP (21), but it is not known whether the bacteria, host, or both produce ATP during infection. In their function as extracellular mediators, nucleotides activate P2 nucleotide receptors that include the ionotrophic P2X receptors (P2X 1 to P2X 7 ) and G-proteincoupled P2Y receptors (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 to P2Y 14 ) (33). In the urinary tract, extracellular ATP may communicate through P2X and P2Y receptors with different cells, such as uroepithelial cells, inflammatory cells, nerves, and myofibroblasts (5). Evidence that P2 receptors, in particular P2Y receptors, may regulate host immunity and modulate phagocytosis, chemotaxis, and cytokine production is now accumulating (14, 16). The P2Y receptor subtypes P2Y 1 , P2Y 2 , and P2Y 4 are expressed throughout the cat bladder urothelium (7), but P2Y receptor expression has not been examined in the human urothelium.Innate immunity is the first line of defense in the urinary tract and triggered by uropathogenic bacteria through activation of Toll-like receptors (TLRs) located on the uroepithelial cell surface (32). Lipopolysaccharide (LPS) is an established ligand for Toll-like receptor 4 (TLR4), but associated coeffector proteins, such as CD14 and MD2, are needed for LPS-induced stimulation of TLR4 (4, 27). TLR4 and CD14 expression by the human urinary tract epithelium remains controversial (3,23,24), and in vitro studies show that the TLR4 receptor can be activated in a LPS-independent manner by P-fimbriated uropathogenic Escherichia coli (UPEC) (12). Once activated, the uroepithelial cells play a central role in the host proinflammatory response through production of chemotactic substances, such as interleukin 6 (IL-6) and IL-8. Several lines of evidence demonstrate that IL-8 is essential for neutrophil transmigration across the infected urothelium and that neutrophils are important for clearance of bacterial infections in the urinary tract (32).

Interleukin-8 and 6 release after purinergic stimulation in uroepithelial A498 cells is mediated through P2Y(2) receptor activation.

Background/Aims: Increased nitric oxide (NO) production or inducible form of NO synthase activity have been documented in patients suffering from urinary tract infection (UTI), but the role of NO in this infection is unclear. We investigated whether NO can affect the host response in human renal epithelial cells by modulating IL-6 production and mRNA expression. Methods: The human renal epithelial cell line A498 was infected with a uropathogenic Escherichia coli (UPEC) strain and/or the NO donor DETA/NO. The IL-6 production and mRNA expression were evaluated by ELISA and real-time RT-PCR. IL-6 mRNA stability was evaluated by analyzing mRNA degradation by real-time RT-PCR. Results: DETA/NO caused a significant (p < 0.05) increase in IL-6 production. Inhibitors of p38 MAPK and ERK1/2 signaling, but not JNK, were shown to significantly suppress DETA/NO-induced IL-6 production. UPEC-induced IL-6 production was further increased (by 73 ± 23%, p < 0.05) in the presence of DETA/NO. The IL-6 mRNA expression increased 2.1 ± 0.17-fold in response to DETA/NO, while the UPEC-evoked increase was pronounced (20 ± 4.5-fold). A synergistic effect of DETA/NO on UPEC-induced IL-6 expression was found (33 ± 7.2-fold increase).The IL-6 mRNA stability studies showed that DETA/NO partially attenuated UPEC-induced degradation of IL-6 mRNA. Conclusions: NO was found to stimulate IL-6 in renal epithelial cells through p38 MAPK and ERK1/2 signaling pathways and also to increase IL-6 mRNA stability in UPEC-infected cells. This study proposes a new role for NO in the host response during UTI by modulating the transcription and production of the cytokine IL-6.

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AimHere we investigated the role of complement activation in phagocytosis and the release of cytokines and chemokines in response to two clinical isolates: Borrelia afzelii K78, which is resistant to complement-mediated lysis, and Borrelia garinii LU59, which is complement-sensitive.Methods Borrelia spirochetes were incubated in hirudin plasma, or hirudin-anticoagulated whole blood. Complement activation was measured as the generation of C3a and sC5b-9. Binding of the complement components C3, factor H, C4, and C4BP to the bacterial surfaces was analyzed. The importance of complement activation on phagocytosis, and on the release of cytokines and chemokines, was investigated using inhibitors acting at different levels of the complement cascade.Results1) Borrelia garinii LU59 induced significantly higher complement activation than did Borrelia afzelii K78. 2) Borrelia afzelii K78 recruited higher amounts of factor H resulting in significantly lower C3 binding. 3) Both Borrelia strains were efficiently phagocytized by granulocytes and monocytes, with substantial inhibition by complement blockade at the levels of C3 and C5. 4) The release of the pro-inflammatory cytokines and chemokines IL-1β, IL-6, TNF, CCL20, and CXCL8, together with the anti-inflammatory IL-10, were increased the most (by>10-fold after exposure to Borrelia). 5) Both strains induced a similar release of cytokines and chemokines, which in contrast to the phagocytosis, was almost totally unaffected by complement blockade.ConclusionsOur results show that complement activation plays an important role in the process of phagocytosis but not in the subsequent cytokine release in response to live Borrelia spirochetes.

Aims: Extracellular ATP may be metabolized to AMP and adenosine by the ectonucleotidases CD39 and CD73 and, in this study, we characterized the pathways for adenosine formation in human urinary tract epithelial cells. Methods: Bladder (RT4) and kidney (A498) epithelial cells were grown in cell culture and the expression of CD39 and CD73 was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. High-performance liquid chromatography was used to determine adenosine formation in cell medium. Results: RT-PCR and immunohistochemistry revealed a high CD73 and a low CD39 expression in human urinary tract epithelial cells, whereas neutrophils had a higher CD39 than CD73 expression. Adenosine was produced when the cells were exposed to 5′-AMP (substrate for CD73), but not when exposed to 5′-ATP (substrate for CD39). A pronounced inhibition of 5′-AMP-induced adenosine formation by the CD73 inhibitor AMP-CP confirmed the involvement of CD73. Adenosine production from 5′-ATP was slightly increased (p < 0.05) when epithelial cells were cocultured with neutrophils. Conclusions: The data demonstrate that adenosine formation from extracellular ATP is negligible in urinary tract epithelial cells due to low CD39 expression in this cell type. However, the epithelial cells express CD73 and are able to convert extracellular AMP to adenosine.

Aims: Adenosine A_2A and A_2B receptor subtypes have both been implicated in the modulation of inflammation. We examined adenosine A_2A and A_2B receptor expression, signaling pathways and the effect of adenosine A_2A and A_2B receptor activation on the uropathogenic Escherichia coli (UPEC)-stimulated IL-8 response in human uroepithelial cells (UROtsa). Methods: Receptor expression was examined by RT-PCR and Western blot, and IL-8 production, intracellular cAMP levels and phosphoproteins were measured by ELISA, EIA and multiplex immunoassay, respectively. Results: The adenosine A₁, A_2A and A_2B receptor subtypes were detected in UROtsa cells. The adenosine A_2A receptor agonist CGS 21680 did not stimulate cAMP production but CREB phosphorylation was slightly increased. The adenosine A₂ receptor agonist CPCA induced a pronounced cAMP and CREB response. Furthermore, CGS 21680 but not CPCA decreased ERK 1/2 and STAT3 phosphorylation. UPEC infection stimulated the host IL-8 production but CPCA or CGS 21680 did not affect UPEC-evoked IL-8 production. Conclusions: Our data identified differences in signaling pathways evoked by adenosine A_2A and A_2B receptor activation. Activation of the adenosine A_2A receptor inhibited STAT3 and ERK 1/2 phosphorylation, while the cAMP-CREB pathway was induced by adenosine A_2B receptor activation. No anti- or proinflammatory effects were found for uroepithelial adenosine A_2A or A_2B receptors.