Urinary tract infection : pathogenesis and complications

Urinary tract infections (UTI) are one of the most common infections in women and children worldwide. If not diagnosed and treated appropriately, it may lead to severe illness and long-term complications. The economic impact caused by UTI on society is significant. In addition, bacterial resistance to common antibiotics is spreading at a high rate. Recent years, the understanding of the host-pathogen interaction and activation of the immune response in the urinary tract has increased considerably. However, there is still a lack of understanding how these basic molecular events can be translated into clinical practise. In this thesis, the complex interaction between the uropathogenic E. coli and the urinary bladder and the kidney epithelium is described in the context of novel findings about virulence factors, immune response and complications following UTI. In the first study, we investigated the chemokine profile in the kidneys of mice with pyelonephritis and the impact of IL-1β by using an IL-1β knockout mouse and renal cell lines. We could show a robust induction of IL-8/MIP-2 and of the chemokines MCP-1 and RANTES, not previously associated with bacterial infection. In the second study, we described a new set of molecules involved in UTI pathogenicity, the metalloproteinase MMP-9 and its natural inhibitor TIMP-1. They increased in experimental pyelonephritis in mice and were found to be expressed by immune cells and resident renal cells. In children with pyelonephritis, we could link TIMP-1 to an increased risk of renal scarring. In the third study, we investigated the virulence factor cytotoxic necrotizing factor (CNF1), commonly found in uropathogenic E. coli. Increased inflammatory reaction could be attributed to CNF1 in vitro; however, urine from patients with UTI caused by CNF1 positive or negative E. coli showed no difference in inflammatory response. Thus, the difference seen in vitro is probably of minor importance in vivo in comparison to other virulence factors of the uropathogenic E. coli. In the last paper, we demonstrated that women supplemented with vitamin D had a stronger response of the antimicrobial peptide cathelicidin when biopsies from the urinary bladder were infected ex vivo. We also showed that normal urinary bladder cells were able to activate 25 hydroxyvitamin D3 to its functional form 1,25 dihydroxyvitamin D3, indicating a role for locally produced vitamin D in the urinary bladder. In addition, bladder cells increased their cathelicidin stores after vitamin D treatment and exerted antibacterial properties against uropathogenic E. coli. In conclusion, this thesis presents new findings about host-microbe interaction in the urinary tract, the immune response and post-infectious complications of UTI. MMP-9, TIMP-1 and the chemokines IL-8/MIP-2, MCP-1 and RANTES were produced in the kidneys during pyelonephritis. IL-1β and TIMP-1 affected the severity of infection and renal scarring. The E. coli toxin CNF1 influenced the immune response only in vitro, but did not seem to influence inflammation in vivo. Finally, treatment with vitamin D increased the antibacterial properties of the uroepithelium by induction of the antimicrobial peptide cathelicidin

Vitamin D Induction of the Human Antimicrobial Peptide Cathelicidin in the Urinary Bladder

The urinary tract is frequently being exposed to potential pathogens and rapid defence mechanisms are therefore needed. Cathelicidin, a human antimicrobial peptide is expressed and secreted by bladder epithelial cells and protects the urinary tract from infection. Here we show that vitamin D can induce cathelicidin in the urinary bladder. We analyzed bladder tissue from postmenopausal women for expression of cathelicidin, before and after a three-month period of supplementation with 25-hydroxyvitamin D3 (25D3). Cell culture experiments were performed to elucidate the mechanisms for cathelicidin induction. We observed that, vitamin D per se did not up-regulate cathelicidin in serum or in bladder tissue of the women in this study. However, when the bladder biopsies were infected with uropathogenic E. coli (UPEC), a significant increase in cathelicidin expression was observed after 25D3 supplementation. This observation was confirmed in human bladder cell lines, even though here, cathelicidin induction occurred irrespectively of infection. Vitamin D treated bladder cells exerted an increased antibacterial effect against UPEC and colocalization to cathelicidin indicated the relevance of this peptide. In the light of the rapidly growing problem of resistance to common urinary tract antibiotics, we suggest that vitamin D may be a potential complement in the prevention of UTI

Vitamin D-deficient mice have more invasive urinary tract infection

<div><p>Vitamin D deficiency is a common health problem with consequences not limited to bone and calcium hemostasis. Low levels have also been linked to tuberculosis and other respiratory infections as well as autoimmune diseases. We have previously shown that supplementation with vitamin D can induce the antimicrobial peptide cathelicidin during <i>ex vivo</i> infection of human urinary bladder. In rodents, however, cathelicidin expression is not linked to vitamin D and therefore this vitamin D-related effect fighting bacterial invasion is not relevant. To determine if vitamin D had further protective mechanisms during urinary tract infections, we therefore used a mouse model. In vitamin D-deficient mice, we detected more intracellular bacterial communities in the urinary bladder, higher degree of bacterial spread to the upper urinary tract and a skewed cytokine response. Furthermore, we show that the vitamin D receptor was upregulated in the urinary bladder and translocated into the cell nucleus after <i>E</i>. <i>coli</i> infection. This study supports a more general role for vitamin D as a local immune response mediator in the urinary tract.</p></div

Vitamin D has a protective effect on the cytoskeletal reorganization caused by <i>E</i>. <i>coli</i> infection of human uroepithelial cells.

<p>The actin filament system of human uroepithelial cells was visualized with TRITC-labelled phalloidin. <i>E</i>. <i>coli</i> strain CFT073 infection caused the formation of cortical bundles of actin filaments (A, arrowheads). Image analysis shows that treatment with <i>E</i>. <i>coli</i> significantly altered cell morphology from control as measured by eccentricity (B). Hence, treatment of infected cells with vitamin D significantly recovered the wild-type phenotype while treatment with vitamin D in the absence of infection also showed wild-type cellular morphology and significant differences from infected phenotype.</p

Higher bacterial burden in dietary-induced vitamin D deficiency.

<p>C57BL/6 mice were fed a vitamin D-deficient diet or control diet (n = 26 for each group) for 7 weeks directly after weaning. After 7 weeks mean serum levels were 4 nM in the deficient diet group vs. 96.7 nM in control mice (A). No difference in weight between the two groups was observed (n = 26 respectively). (B). More bacteria spread to the upper urinary tract in the vitamin D-deficient group as measured by bacterial count in the kidneys, (n = 9 in each group) (C) whereas no difference in bacterial load in the urinary bladder of the vitamin D-deficient and supplemented mice was observed, (n = 7 vitamin D deficient mice; n = 8 control mice) (D).</p

Human bladder cells show nuclear translocation and upregulation of VDR upon uropathogenic <i>E</i>. <i>coli</i> infection.

<p>To confirm that VDR conveys vitamin D signaling in the human urinary bladder, VDR silencing with siRNA was performed in bladder epithelial cells, TERT-NHUC. The expression of the vitamin D downstream target <i>CYP24A1</i> was almost completely abolished (A). Two independent experiments. Bladder cells were then infected with <i>E</i>. <i>coli</i> CFT073 for 45 (B), 90 (C) and 120 minutes (D). VDR increased and translocated into the nucleus at 90 minutes. Infected cells without primary antibody served as controls (inserted in B). Further, bladder cells were transfected with VDRE firefly constructs. In the presence of physiological levels of vitamin D, uropathogenic <i>E</i>. <i>coli</i> increased the transcriptional activity of VDR compared to uninfected cells. Pooled values from two independent experiments (n = 7 with 3 and 4 analyses in the respective experiments).</p

Vitamin D receptor (VDR) increases over time following <i>E</i>. <i>coli</i> infection of the uroeptithelium.

<p>C57BL/6 mice were infected with uropathogenic <i>E</i>. <i>coli</i> CFT073 for 14 (A), 24 (B) and 48 hours (C). Staining for VDR protein shows a time-dependent upregulation with initial patchy staining and progressive accumulation. Section without primary antibody is shown as control (D). Bladder lumen is marked with *. Four mice for each time-point.</p

Altered bacterial invasion in vitamin D-deficient bladders.

<p>Uropathogenic <i>E</i>. <i>coli</i> strain CFT073 (red) form aggregates in vitamin D-deficient bladders both in the form of intracellular bacterial communities, IBC (upper panel, left and center, arrow) and large elongated bacterial complexes (upper panel right, arrow) not seen in the vitamin D-sufficient control mice (lower panel). Although also heavily infected, bacteria tended to respect the epithelial border lined with uroplakin (green) (n = 4 in each group). Three representative pictures from four vitamin D deficient and four vitamin D sufficient, control mice (Nuclei are stained with DAPI (blue). Objective used x 63.</p