Mycobacterium bovis bacilli Calmette-Guerin regulates leukocyte recruitment by modulating alveolar inflammatory responses.

Leukocyte migration into the epithelial compartment is an important feature in the active phase of mycobacterial infections. In this study, we used the Transwell model to investigate the mechanisms behind mycobacteria-induced leukocyte recruitment and investigated the role of TLR2 and TLR4 in this process. Infection of epithelial cells resulted in significantly increased secretion of the neutrophil chemotactic CXCL8 and IL-6, but no secretion of monocyte chemotactic CCL2 or TNF-α was observed. In contrast to epithelial response, mycobacteria-infected neutrophils and monocytes secreted all these cytokines. Corresponding with epithelial cytokine response, mycobacterial infection of the epithelial cells increased neutrophil diapedesis, but decreased monocyte recruitment. However, monocyte recruitment towards mycobacteria infected epithelial cells significantly increased following addition of neutrophil pre-conditioned medium. Mycobacterial infection also increases alveolar epithelial expression of TLR2, but not TLR4, as analyzed by flow cytometry, Western blotting and visualized by confocal microscopy. Blocking of TLR2 inhibited neutrophil recruitment and cytokine secretion, while blocking of TLR4 had a lesser effect. To summarize, we found that primary alveolar epithelial cells produced a selective TLR2-dependent cytokine secretion upon mycobacterial infection. Furthermore, we found that cooperation between cells of the innate immunity is required in mounting proper antimicrobial defence

Acute pyelonephritis and renal scarring are caused by dysfunctional innate immunity in mCxcr2 heterozygous mice

The CXCR1 receptor and chemokine CXCL8 (IL-8) support neutrophil-dependent clearance of uropathogenic Escherichia coli from the urinary tract. CXCR1 is reduced in children prone to pyelonephritis, and heterozygous hCXCR1 polymorphisms are more common in this patient group than in healthy individuals, strongly suggesting a disease association. Since murine CXCR2 (mCXCR2) is functionally similar to human CXCR1, we determined effects of gene heterozygosity on the susceptibility to urinary tract infection by infecting heterozygous (mCxcr2+/−) mice with uropathogenic Escherichia coli. Clearance of infection and tissue damage were assessed as a function of innate immunity in comparison to that in knockout (mCxcr2−/−) and wild-type (mCxcr2+/+) mice. Acute sepsis-associated mortality was increased and bacterial clearance drastically impaired in heterozygous compared to wild-type mice. Chemokine and neutrophil responses were delayed along with evidence of neutrophil retention and unresolved kidney inflammation 1 month after infection. This was accompanied by epithelial proliferation and subepithelial fibrosis. The heterozygous phenotype was intermediate, between knockout and wild-type mice, but specific immune cell infiltrates that accompany chronic infection in knockout mice were not found. Hence, the known heterozygous CXCR1 polymorphisms may predispose patients to acute pyelonephritis and urosepsis

Mucosal receptors involved in pathogen recognition and host defence

Urinary tract infection is the most common infection in man. P fimbriae are virulence factors of uro-pathogenic Escherichia coli. They are expressed by 70-90 % of acute pyelonephritis isolates and up to 100% of the most virulent isolates that cause bacteremia. This thesis concerns the interaction between P fimbriae and the urinary tract mucosa, and the host receptors involved in fimbrial recognition and trans membrane signalling. 1. Recognition receptors. P fimbriae recognise receptor epitopes in cell surface glycosphingolipids. When the receptor expression was inhibited with the synthetic ceramide analogue PDMP, attachment was inhibited, as was the cellular response to infection, demonstrating that these receptors are essential for the initial tissue attack by the bacteria. Similar results were obtained using the natural iminosugar NB-DNJ, which blocks the synthesis of ceramide-linked oligosaccharides. In vivo inhibition of the UDP-glucose-glycosyltransferase was shown to cause receptor depletion on the uroepithelial cells and the P fimbriated E. coli failed to colonize the treated mice or to evoke an inflammatory response. The results confirmed the importance of glycosphingolipid receptors for fimbriae, and suggested that NB-DNJ might be explored as a therapeutic alternative in the treatment of UTI. 2. TLR4 and the innate host response. Transmembrane signalling in response to P fimbriated E. coli was shown to depend on TLR4. TLR4 KO mice became chronically infected, and were unable to clear an experimental infection. The results showed that a single host gene can determine the quality of the mucosal response and that this response determines if infection will become symptomatic or asymptomatic. 3. Adaptor protein usage. Two groups of adaptor proteins are involved in TLR4 signalling. The MyD88 /TIRAP adaptors control the CD14 dependent response to LPS/LBP, while TRIF/TRAM have other functions. This study showed that trif/tram controls the CD14 independent cell activation by P fimbriated E. coli. Mice lacking these adaptors developed acute symptoms and were unable to clear the infection. The LPS related Myd88 and TIRAP adaptors, in contrast, were not required to activate the innate defence, but they were needed for efficient clearance of infection. Defects in the TRIF/TRAM pathway may explain the tendency of certain individuals to develop asympromatic bacteriurea rather than symptomatic infection. 4. Ceramide as a signalling intermediate. Following the binding of P fimbriae, the glycosphingolipid receptors are cleaved and ceramide is released. Ceramide has previously been recognised as a signalling intermediate in the sphingomyelin pathway. The results of this study demonstrate that ceramide can act as a signalling intermediate between the recognition receptor and TLR4, thus providing a mechanism for the recruitment of TLR4 by P fimbriated bacteria. 5. Renal scarring in mIL8Rh KO mice. Experimental UTI in mIL-8Rh deficient mice identified the first genetic defect explaining the increased susceptibility to acute pyelonephritis and renal scarring in certain individuals. The IL-8 receptor deficiency caused massive neutrophil trapping in the kidney with tissue damage resembling renal scarring. The mIL-8Rh deficient mouse thus offers a highly relevant model to study the cellular and molecular mechanisms that underlie the development of renal scarring

Glycolipid receptor depletion as an approach to specific antimicrobial therapy.

Mucosal pathogens recognize glycoconjugate receptors at the site of infection, and attachment is an essential first step in disease pathogenesis. Inhibition of attachment may prevent disease, and several approaches have been explored. This review discusses the prevention of bacterial attachment and disease by agents that modify the glycosylation of cell surface glycoconjugates. Glycosylation inhibitors were tested in the urinary tract infection model, where P-fimbriated Escherichia coli rely on glycosphingolipid receptors for attachment and tissue attack. N-butyldeoxynojirimycin blocked the expression of glucosylceramide-derived glycosphingolipids and attachment was reduced. Bacterial persistence in the kidneys was impaired and the inflammatory response was abrogated. N-butyldeoxynojirimycin was inactive against strains which failed to engage these receptors, including type 1 fimbriated or nonadhesive strains. In vivo attachment has been successfully prevented by soluble receptor analogues, but there is little clinical experience of such inhibitors. Large-scale synthesis of complex carbohydrates, which could be used as attachment inhibitors, remains a technical challenge. Antibodies to bacterial lectins involved in attachment may be efficient inhibitors, and fimbrial vaccines have been developed. Glycosylation inhibitors have been shown to be safe and efficient in patients with lipid storage disease and might therefore be tested in urinary tract infection. This approach differs from current therapies, including antibiotics, in that it targets the pathogens which recognize these receptors

Bacterial Adherence and Epithelial Cell Cytokine Production

Epithelial cell lines produce the cytokines IL-1α, IL-6 and IL-8 when stimulated with Escherichia coli. The cytokine response is enhanced by P fimbriae. The epithelial cell lines also respond to stimulation with other cytokines. These in vitro findings were confirmed in vivo in patients with E. coli infection who secreted IL-6 and IL-8 into the urine. The observations suggest that epithelial cells play a more active role in the mucosal immune response than previously recognized

HAMLET Treatment Delays Bladder Cancer Development.

PURPOSE: HAMLET is a protein-lipid complex that kills different types of cancer cells. Recently we observed a rapid reduction in human bladder cancer size after intravesical HAMLET treatment. In this study we evaluated the therapeutic effect of HAMLET in the mouse MB49 bladder carcinoma model. MATERIALS AND METHODS: Bladder tumors were established by intravesical injection of MB49 cells into poly L-lysine treated bladders of C57BL/6 mice. Treatment groups received repeat intravesical HAMLET instillations and controls received alpha-lactalbumin or phosphate buffer. Effects of HAMLET on tumor size and putative apoptotic effects were analyzed in bladder tissue sections. Whole body imaging was used to study HAMLET distribution in tumor bearing mice compared to healthy bladder tissue. RESULTS: HAMLET caused a dose dependent decrease in MB49 cell viability in vitro. Five intravesical HAMLET instillations significantly decreased tumor size and delayed development in vivo compared to controls. TUNEL staining revealed selective apoptotic effects in tumor areas but not in adjacent healthy bladder tissue. On in vivo imaging Alexa-HAMLET was retained for more than 24 hours in the bladder of tumor bearing mice but not in tumor-free bladders or in tumor bearing mice that received Alexa-alpha-lactalbumin. CONCLUSIONS: Results show that HAMLET is active as a tumoricidal agent and suggest that topical HAMLET administration may delay bladder cancer development

pap genotype and P fimbrial expression in Escherichia coli causing bacteremic and nonbacteremic febrile urinary tract infection

Escherichia coli strains from patients with febrile urinary tract infections (n=73) were examined for pap genotype and P fimbrial expression in relation to bacteremia and patients' background variables. Most isolates were pap(+) by DNA hybridization (n=51), and 36 were papG(IA2)(+) and 18 prsG(J96)(+) by polymerase chain reaction. The pap and papG genotypes of the infecting strain were shown to vary with host compromise, sex, and age. Bacteremia in noncompromised patients was caused by papG(IA2)(+) strains, but compromised hosts carried a mixture of papG(IA2)(+), prsG(J96)(+), and pap(-) strains. Women of all ages were infected with papG(IA2)(+) strains. Infected men carried prsG(J96)(+) or pap(-) strains and were older, and most had compromising conditions. papG(IA2)(+) strains predominated among patients with medical illness, whereas prsG(J96)(+) strains predominated among patients with urinary tract abnormalities. These findings emphasize the strong influence of host factors on the selection of E. coli strains causing febrile urinary tract infections

Role of the ceramide signalling pathway in cytokine responses to P fimbriated Escherichia coli.

Escherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response. The receptors for E. coli P fimbriae are the globoseries of glycosphingolipids; Gal alpha 1-->4Gal beta-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer. The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids. This study tested the hypothesis that P-fimbriated E. coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell. We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls. P-fimbriated E. coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate. The IL-6 response to P-fimbriated E. coli was reduced by inhibitors of serine/threonine kinases but not by other protein kinase inhibitors. In contrast, ceramide levels were not influenced by type 1-fimbriated E. coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors. These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E. coli, and that the receptor specificity of the P fimbriae influences this process. We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E. coli in epithelial cells

Strategies for studying bacterial adhesion in Vivo

The ultimate goal of studies on microbial adhesion is to understand what molecular interactions between the host and microbe occur in vivo and the impact of these interactions on disease processes. With this goal in mind, the problem can be approached at four levels. At the biochemical level, the host receptors at the relevant colonization site are identified; at the cell biology level, consequences of bacterial binding to host epithelial cells are studied in cell culture; at the physiological level, the consequences of bacterial binding are studied in experimental animals or humans; and at the population level, the consequences of receptor binding for colonization are studied by epidemiological methods. This chapter provides an example of studies at each level and discusses the implications for what might occur in vivo

The role of lipopolysaccharide and Shiga-like toxin in a mouse model of Escherichia coli O157:H7 infection

The role of lipopolysaccharide (LPS) and Shiga-like toxin (SLT) in the pathogenesis of hemolytic uremic syndrome (HUS) was studied in a mouse model. Mice inoculated intragastrically with Escherichia coli O157:H7 developed gastrointestinal, neurologic, and systemic symptoms, necrotic foci in the colon, glomerular and tubular histopathology, and fragmented erythrocytes. LPS-responder (C3H/HeN) mice developed a combination of neurologic and systemic symptoms, whereas LPS-nonresponder (C3H/HeJ) mice had a biphasic course of disease, first developing systemic symptoms and later severe neurologic symptoms. Mice inoculated with SLT-II-positive strains developed severe neurotoxic symptoms and a higher frequency of systemic symptoms and glomerular pathology compared with SLT-II-negative strains. Anti-SLT-II antibodies protected against these symptoms and pathology. These results demonstrate that this model could be used to study aspects of human HUS and that both LPS and SLT are important for disease development