Maria Hedlund

Introduction to galectins.

Good evidence suggest roles of galectins in cancer, immunity and inflammation, and development, but a unifying picture of their biological function is lacking. Instead galectins appear to have a particularly diverse, bewildering but intriguing array of activities both inside and outside cells—“clear truths and mysteries are inextricably twined”. Fortunately this has not discouraged but rather enthused a large number of good galectin researchers, some of which have contributed to this special issue of Glycoconjugate Journal to provide a personal, critical status of the field. Here we will give a brief introduction to the galectins as a protein family with some comments on nomenclature. Published in 2004.

Cytokine responses during mucosal infections: role in disease pathogenesis and host defence

Mucosal pathogens use diverse and highly specific molecular mechanisms to activate mucosal inflammation. It may even be argued that their virulence depends on the inflammatory response that they induce. Some bacteria target epithelial cells and trigger them to produce inflammatory mediators but others cross the mucosa and activate macrophages or dendritic cells. Although systemic release of inflammatory mediators causes many symptoms and signs of infection, local chemokine production leads to the recruitment of inflammatory cells and lymphocytes that participate directly in the clearance of bacteria from mucosal sites. In this way, mucosal inflammation is a two-edged sword responsible for disease associated tissue destruction and crucial for the antimicrobial defence. Understanding of these pathways should create tools to enhance the defence and interfere with disease.

Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation

Fimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae‐dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll‐like receptor 4 (TLR4)‐dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS‐CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co‐staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co‐localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4‐dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co‐receptors.

Type 1 fimbriae deliver an LPS- and TLR4-dependent activation signal to CD14-negative cells

Fimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae‐dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll‐like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS‐ and TLR4‐dependent signal to the CD14‐negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS‐dependent response in those cells. A similar LPS‐ and fimbriae‐dependent response was observed in the urinary tract of TLR4‐proficient mice, but not in TLR4‐defective mice. The moderate inflammatory response in the TLR4‐defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14‐negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host ‘sees’ LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells.

P FIMBRIAE-DEPENDENT, LIPOPOLYSACCHARIDE-INDEPENDENT ACTIVATION OF EPITHELIAL CYTOKINE RESPONSES

Cells in the mucosal barrier are equipped to sense and respond to microbes in the lumen and translate this molecular information into signals that can reach local or distant sites. The interaction of P‐fimbriated Escherichia coli with human uroepithelial cells is a model to study the molecular mechanism of epithelial cell activation by mucosal pathogens. Here, we examine the role of lipopolysaccharide (LPS) as a co‐stimulatory molecule in epithelial cell activation by P‐fimbriated E. coli. P‐fimbriated clinical isolates or recombinant strains were shown to trigger a fimbriae‐dependent epithelial cell cytokine response. Mutational inactivation of the msbB sequences that control lipid A myristoylation drastically impaired monocyte stimulation but not epithelial responses to P‐fimbriated bacteria. Polymyxin B or bactericidal/permeability increasing factor (BPI) neutralized the effects of lipid A in the monocyte assay, but did not reduce epithelial responses. Finally, isolated LPS of the smooth, rough and deep rough chemotypes were poor epithelial cell activators. The cells were shown to lack surface CD14 or CD14 mRNA as well as the CD14 co‐receptor function and were also very poor LPS responders in the presence of human serum. These results demonstrate that epithelial cell responses to P‐fimbriated E. coli are CD14 and LPS independent, and suggest that attaching pathogens can overcome the LPS unresponsiveness of epithelial cells by fimbriae‐dependent activation mechanisms.

Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P-fimbriated Escherichia coli

Uropathogenic Escherichia coli attach to epithelial cells through P fimbriae that bind Galα1‐4Galβ‐oligosaccharide sequences in cell surface glycosphingolipids. The binding of P‐fimbriated E. coli to uroepithelial cells causes the release of ceramide, activation of the ceramide signalling pathway and a cytokine response in the epithelial cells. The present study examined the molecular source of ceramide in human kidney A498 cells exposed to P‐fimbriated E. coli. Agonists such as TNF‐α and IL‐1β released ceramide from sphingomyelin by the activation of endogenous sphingomyelinases and hydrolysis of sphingomyelin, and triggered an IL‐6 response. P‐fimbriated E. coli caused a slight increase in endogenous sphingomyelinase activity, but there was no associated sphingomyelin hydrolysis. Instead, the concentration of galactose‐containing glycolipids decreased. We propose that P‐fimbriated E. coli differ from other activators of the ceramide pathway, in that release of ceramide is from receptor glycolipids and not from sphingomyelin. Receptor breakdown may be an efficient host defence strategy, as it reduces the concentration of cell surface receptors, releases soluble receptor analogues and activates an inflammatory response.

Toll-like Receptor Signaling and Chemokine Receptor Expression Influence the Severity of Urinary Tract Infection

Urinary tract infections (UTIs) vary in pathogenesis and severity. After their ascent into the urinary tract, bacteria may establish asymptomatic bacteriuria (ABU), cause acute cystitis, or cause acute pyelonephritis. Research during the last few decades has established that the site of infection and the disease severity are influenced by bacterial virulence. In the 1940s, hemolysin was shown to identify Escherichia coli that cause extraintestinal infections [1]. “Uropathogenic” E. coli strains were later shown to belong to a restricted set of serotypes or clones [2], and acute pyelonephritis and ABU strains were shown to differ in surface antigen repertoire [3]. Studies in the 1970s started to involve host cell interactions with attachment to the urinary tract mucosa [4]. We proposed that the disease severity was a direct result of bacterial virulence and that tissue attachment is a first critical step. The special virulence of the uropathogenic clones has subsequently been shown to include numerous virulence factors encoded on the pathogenicity islands (see Middendorf et al., this issue). The variation in urinary tract virulence reflects the ability of bacteria to trigger mucosal and systemic host responses. Through different molecular interactions, bacteria may trigger epithelial cell responses, cause cell detachment, and invade or kill cells by apoptosis (for review see [5]). Inflammation has received special attention because it determines the severity of UTI and the clearance of infection [6] (also see Agace et al. in [5]). We have studied how the inflammatory response is initiated and how it determines the resistance to UTI. Herein we argue that individuals differ in the ability to respond to UTI. We propose that pyelonephritis occurs more readily in “high responders” and that their abnormalities exaggerate the damaging rather than the protective aspects of inflammation. The “low responders,” on the other hand, have suppressed inflammatory signals, allowing bacteriuria to establish without harm

The ‘innate’ host response protects and damages the infected urinary tract

Symptoms of infection and tissue pathology are caused by the host response: not by the microbe per se. The same response is also critical for the defence and is needed to clear infection. It is therefore essential to understand how the host response is activated and to identify the critical effector mechanisms of the defence. We have studied these issues in the urinary tract infection (UTI) model. The symptoms of UTI and the host defence both rely on the so-called ‘innate’ immune system, making this one of the best characterized human disease models of ‘innate immunity’. We discuss the critical molecular events that determine whether the host response will be activated by P-fimbriated uropathogenic Escherichio coli as well as factors determining whether the patient develops acute pyelonephritis or asymptomatic bacteriuria. We will describe the glycoconjugate receptors used by the P-fimbriated bacteria adhering to host tissues, the recruitment of TLR4 co-receptors and the signalling pathways that allow progression to symptomatic disease, and discuss how these mechanisms are altered in asymptomatic carriers, presenting the possible genetic basis for unresponsiveness. We have shown that neutrophils are the critical effectors of the host defence and that neutrophil dysfunctions lead to acute pyelonephritis and renal scarring. Here we discuss the mechanisms of neutrophil-mediated, chemokine receptor (CXCR1)-dependent clearance, and the defect in inter-leukin-8 receptor homolog knock-out (IL-8Rh KO) mice and describe the data linking low CXCRI expression to recurrent pyelonephritis in man, as well as the information on the genetic basis for low CXCR1 expression in affected patients. Finally, the mechanisms of renal scarring in IL8Rh KO mice will be discussed in relation to human disease. Our studies hold the promise to provide a molecular and genetic explanation for disease susceptibility in some patients with UTI and to offer more precise tools for the diagnosis and therapy of these infections.

Fimbriae, Transmembrane Signaling, and Cell Activation

Attachment to mucosal surfaces enables commensal organisms to select their ecologic niche and pathogens to select their site of infection. The tissue-specific attachment involves the coupling of microbial surface adhesins to receptor epitopes on host cell surfaces and may occur in competition with soluble receptor molecules. Gram-negative bacteria carry fimbriae with lectinlike domains that recognize receptor epitopes in cell surface glycolipids or glycoproteins. The resulting specific adherence may enhance bacterial persistence at the site of infection, but adherence also has been identified as a virulence factor that facilitates tissue attack, invasion, and mucosal inflammation. Two general mechanisms have been proposed to explain how attachment promotes tissue attack by pathogenic bacteria. In the first case, the coupling of fimbriae to their receptors activates receptor-determined signaling pathways in the host cell

Bacterial Attachment To Uro-Epithelial Cells: Mechanisms and Consequences

Microbial attachment to mucosal surfaces is a first step in mucosal infection. Specific interactions between microbial surface ligands and host receptors influence the distribution of microbes in their sites of infection. Adhesion has often been regarded as a sufficient end point, explaining tissue tropism and bacterial persistence at mucosal sites. Adherence, however, is also a virulence factor through which microbes gain access to host tissues, upset the integrity of the mucosal barrier, and cause disease. The induction of mucosal inflammation is one aspect of this process. Bacterial attachment to mucosal surfaces activates the production of pro-inflammatory cytokines that cause both local and systemic inflammation. Epithelial cells are one source of these cytokines. The binding of fimbrial lectins to epithelial cell receptors triggers transmembrane signaling events that upregulate cytokine-specific mRNA and increase cytokine secretion. P fimbriae that bind the globoseries of glycolipids cause the release of ceramides and activation of the ceramide signaling pathway which contributes to the IL-6 response. Spread of cytokines and other pro-inflammatory mediators from the local site contributes to the symptoms and signs of infection.