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1.  The combined role of galactose-deficient IgA1and streptococcal IgA-binding M protein in inducing IL-6 and C3 secretion from human mesangial cells: implications for IgA nephropathy 
IgA nephropathy is characterized by mesangial cell proliferation and extracellular matrix expansion associated with immune deposits consisting of galactose-deficient polymeric IgA1 and C3. We have previously shown that IgA-binding regions of streptococcal M proteins co-localize with IgA in mesangial immune deposits in patients with IgA nephropathy. In the current study, the IgA-binding M4 protein from group A streptococcus was found to bind to galactose-deficient polymeric IgA1 with higher affinity than to other forms of IgA1, as shown by surface plasmon resonance and solid-phase immunoassay. The M4 protein was demonstrated to bind to mesangial cells not via the IgA-binding region but rather via the C-terminal region, as demonstrated by flow cytometry. IgA1 enhanced binding of M4 to mesangial cells, but not vice versa. Co-stimulation of human mesangial cells with M4 and galactose-deficient polymeric IgA1 resulted in a significant increase in IL-6 secretion compared to each stimulant alone. Galactose-deficient polymeric IgA1 alone, but not M4, induced C3 secretion from the cells and co-stimulation enhanced this effect. In addition, co-stimulation enhanced mesangial cell proliferation compared to each stimulant alone. These results indicate that IgA-binding M4 protein binds preferentially to galactose-deficient polymeric IgA1 and that these proteins together induce excessive pro-inflammatory responses and proliferation of human mesangial cells. Thus, tissue deposition of streptococcal IgA-binding M proteins may contribute to the pathogenesis of IgA nephropathy.
PMCID: PMC4065838  PMID: 24850720
2.  A Novel Mechanism of Bacterial Toxin Transfer within Host Blood Cell-Derived Microvesicles 
PLoS Pathogens  2015;11(2):e1004619.
Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.
Author Summary
Shiga toxin-producing enterohemorrhagic Escherichia coli are non-invasive bacteria that, after ingestion, cause disease by systemic release of toxins and other virulence factors. These infections cause high morbidity, including hemolytic uremic syndrome with severe anemia, low platelet counts, renal failure, and mortality. The most common clinical isolate is E. coli O157:H7. In 2011 an E. coli O104:H4 strain caused a large outbreak in Europe with high mortality. After Shiga toxin damages intestinal cells it comes in contact with blood cells and thus gains access to the circulation. In this study we have shown that the toxin is released into circulating host blood cell-derived microvesicles, in which it retains its toxicity but evades the host immune response. Our results suggest that these microvesicles can enter target organ cells in the kidney and transfer toxin into these cells as well as between cells. Such a mechanism of virulence has not been previously described in bacterial infection.
PMCID: PMC4342247  PMID: 25719452
3.  Complement activation associated with ADAMTS13 deficiency in human and murine thrombotic microangiopathy 
This study addressed the contribution of ADAMTS13-deficiency to complement activation in thrombotic thrombocytopenic purpura (TTP). Renal tissue and blood samples were available from 12 TTP patients. C3 and C5b-9 deposition were demonstrated in the renal cortex of two TTP patients, by immunofluorescence and immunohistochemistry, respectively. C3 was also demonstrated in the glomeruli of Shiga toxin-2 treated Adamts13−/− mice (n=6 of 7) but less in mice that were not Shiga toxin-2-treated (n=1 of 8, p<0.05) or wild-type mice (n=0 of 7). TTP patient plasma (n=9) contained significantly higher levels of complement-coated endothelial microparticles than control plasma (n=13), as detected by flow cytometry. Exposure of histamine-stimulated primary glomerular endothelial cells to platelet-rich-plasma from patients, or patient platelet-poor-plasma combined with normal platelets, in a perfusion system, under shear, induced C3 deposition on von Willebrand factor (VWF)-platelet strings (on both VWF and platelets) and on endothelial cells. Complement activation occurred via the alternative pathway. No C3 was detected when cells were exposed to TTP plasma that was pre-incubated with EDTA or heat-inactivated, or to control plasma. In the perfusion system patient plasma induced more release of C3- and C9-coated endothelial microparticles compared to control plasma. The results indicate that the microvascular process induced by ADAMTS13 deficiency triggers complement activation on platelets and the endothelium, which may contribute to formation of thrombotic microangiopathy.
PMCID: PMC3750088  PMID: 23878316
4.  Shiga Toxin Pathogenesis: Kidney Complications and Renal Failure 
The kidneys are the major organs affected in diarrhea-associated hemolytic uremic syndrome (D+HUS). The pathophysiology of renal disease in D+HUS is largely the result of the interaction between bacterial virulence factors such as Shiga toxin and lipopolysaccharide and host cells in the kidney and in the blood circulation. This chapter describes in detail the current knowledge of how these bacterial toxins may lead to kidney disease and renal failure. The toxin receptors expressed by specific blood and resident renal cell types are also discussed as are the actions of the toxins on these cells.
PMCID: PMC3779650  PMID: 21983749
5.  The Antimicrobial Peptide Cathelicidin Protects Mice from Escherichia coli O157:H7-Mediated Disease 
PLoS ONE  2012;7(10):e46476.
This study investigated the role of the antimicrobial peptide cathelicidin in Escherichia coli O157:H7 infection and subsequent renal damage. Mouse and human cathelicidin, CRAMP and LL-37, respectively, killed E. coli O157:H7 in vitro. Intestines from healthy wild-type (129/SvJ) and cathelicidin-knock-out (Camp−/−) mice were investigated, showing that cathelicidin-deficient mice had a thinner colonic mucus layer compared with wild-type mice. Wild-type (n = 11) and cathelicidin-knock-out (n = 11) mice were inoculated with E. coli O157:H7. Cathelicidin-deficient animals exhibited higher fecal counts of E. coli O157:H7 and bacteria penetrated the mucus forming attaching-and-effacing lesions to a much higher extent than in wild-type animals. Cathelicidin knock-out mice developed symptoms (9/11) as well as anemia, thrombocytopenia and extensive renal tubular damage while all cathelicidin-producing mice remained asymptomatic with normal laboratory findings. When injected with Shiga toxin intraperitoneally, both murine strains developed the same degree of renal tubular damage and clinical disease indicating that differences in sensitivity to infection between the murine strains were related to the initial intestinal response. In conclusion, cathelicidin substantially influenced the antimicrobial barrier in the mouse colon mucosa. Cathelicidin deficiency lead to increased susceptibility to E. coli O157:H7 infection and subsequent renal damage. Administration of cathelicidin or stimulation of endogenous production may prove to be novel treatments for E. coli O157:H7-induced hemolytic uremic syndrome.
PMCID: PMC3471911  PMID: 23077510
7.  Cross-Reactive Protection against Enterohemorrhagic Escherichia coli Infection by Enteropathogenic E. coli in a Mouse Model ▿  
Infection and Immunity  2011;79(6):2224-2233.
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related attaching and effacing (A/E) pathogens. The genes responsible for the A/E pathology are carried on a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Both pathogens share a high degree of homology in the LEE and additional O islands. EHEC prevalence is much lower in areas where EPEC is endemic. This may be due to the development of antibodies against common EPEC and EHEC antigens. This study investigated the hypothesis that EPEC infections may protect against EHEC infections. We used a mouse model to inoculate BALB/c mice intragastrically, first with EPEC and then with EHEC (E. coli O157:H7). Four control groups received either a nonpathogenic E. coli (NPEC) strain followed by EHEC (NPEC/EHEC), phosphate-buffered saline (PBS) followed by EHEC (PBS/EHEC), EPEC/PBS, or PBS/PBS. Mice were monitored for weight loss and symptoms. EPEC colonized the intestine after challenge, and mice developed serum antibodies to intimin and E. coli secreted protein B (encoded in the LEE). Prechallenge with an EPEC strain had a protective effect after EHEC infection, as only a few mice developed mild symptoms, from which they recovered. These mice had an increase in body weight similar to that in control animals, and tissue morphology exhibited mild intestinal changes and normal renal histology. All mice that were not prechallenged with the EPEC strain developed mild to severe symptoms after EHEC infection, with weight loss as well as intestinal and renal histopathological changes. These data suggest that EPEC may protect against EHEC infection in this mouse model.
PMCID: PMC3125830  PMID: 21402761
8.  Phenotypic Expression of ADAMTS13 in Glomerular Endothelial Cells 
PLoS ONE  2011;6(6):e21587.
ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13+/+) and deficient (Adamts13−/−) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells.
Methodology/Principal Findings
Immunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice. Phenotypic differences were examined by ultramorphology. ADAMTS13 expression in human glomerular endothelial cells and dermal microvascular endothelial cells was investigated by real-time PCR, flow cytometry, immunofluorescence and immunoblotting. VWF cleavage was demonstrated by multimer structure analysis and immunoblotting. ADAMTS13 was demonstrated in glomerular endothelial cells in Adamts13+/+ mice but no staining was visible in tissue from Adamts13−/− mice. Thickening of glomerular capillaries with platelet deposition on the vessel wall was detected in Adamts13−/− mice. ADAMTS13 mRNA and protein were detected in both human endothelial cells and the protease was secreted. ADAMTS13 activity was demonstrated in glomerular endothelial cells as cleavage of VWF.
Glomerular endothelial cells express and secrete ADAMTS13. The proteolytic activity could have a protective effect preventing deposition of platelets along capillary lumina under the conditions of high shear stress present in glomerular capillaries.
PMCID: PMC3123364  PMID: 21720563
9.  Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney Infection 
PLoS Pathogens  2010;6(9):e1001109.
The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3−/− mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3−/− mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response.
Author Summary
The host immune system must identify pathogens and defeat them through TLR-dependent signaling pathway activation, while distinguishing them from commensal flora. Contrary to current dogma, the host cannot solely use “pattern recognition” since the microbial molecules involved in such recognition are present on pathogens and commensals alike. We identify here a pathogen-specific mechanism of TLR4 activation and signaling intermediates in this pathway, leading to IRF3-dependent transcription of innate immune response genes. We show in knockout mice that Irf3 deficiency causes severe tissue pathology and that effector functions controlled by IFNβ are involved. Finally, in highly disease-prone pyelonephritis patients we found a high frequency of IRF3 promoter polymorphism compared to asymptomatic bacterial carriers or controls. The polymorphisms influenced promoter activity in reporter assays, suggesting that they are functionally important. Urinary tract infections are among the most common bacterial infections in man, and are a major cause of morbidity and mortality. A subset of disease-prone individuals is at risk for recurrent disease, severe renal dysfunction and end-stage renal disease. At present, there is no method to identify disease-prone infants and to prevent future morbidity and renal damage. The genetic and functional studies described here indicate that genetic variation in IRF3 influences individual susceptibility to kidney infection and might serve as a new tool for future risk assessment in this patient group.
PMCID: PMC2944801  PMID: 20886096
10.  Toll-Like Receptor 4 Promoter Polymorphisms: Common TLR4 Variants May Protect against Severe Urinary Tract Infection 
PLoS ONE  2010;5(5):e10734.
Polymorphisms affecting Toll-like receptor (TLR) structure appear to be rare, as would be expected due to their essential coordinator role in innate immunity. Here, we assess variation in TLR4 expression, rather than structure, as a mechanism to diversify innate immune responses.
Methodology/Principal Findings
We sequenced the TLR4 promoter (4,3 kb) in Swedish blood donors. Since TLR4 plays a vital role in susceptibility to urinary tract infection (UTI), promoter sequences were obtained from children with mild or severe disease. We performed a case-control study of pediatric patients with asymptomatic bacteriuria (ABU) or those prone to recurrent acute pyelonephritis (APN). Promoter activity of the single SNPs or multiple allelic changes corresponding to the genotype patterns (GPs) was tested. We then conducted a replication study in an independent cohort of adult patients with a history of childhood APN. Last, in vivo effects of the different GPs were examined after therapeutic intravesical inoculation of 19 patients with Escherichia coli 83972. We identified in total eight TLR4 promoter sequence variants in the Swedish control population, forming 19 haplotypes and 29 genotype patterns, some with effects on promoter activity. Compared to symptomatic patients and healthy controls, ABU patients had fewer genotype patterns, and their promoter sequence variants reduced TLR4 expression in response to infection. The ABU associated GPs also reduced innate immune responses in patients who were subjected to therapeutic urinary E. coli tract inoculation.
The results suggest that genetic variation in the TLR4 promoter may be an essential, largely overlooked mechanism to influence TLR4 expression and UTI susceptibility.
PMCID: PMC2873976  PMID: 20505764
11.  Shiga Toxin and Lipopolysaccharide Induce Platelet-Leukocyte Aggregates and Tissue Factor Release, a Thrombotic Mechanism in Hemolytic Uremic Syndrome 
PLoS ONE  2009;4(9):e6990.
Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)–bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF.
Methodology/Principal Findings
The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF–expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery.
By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.
PMCID: PMC2735777  PMID: 19750223
12.  A Genetic Basis of Susceptibility to Acute Pyelonephritis 
PLoS ONE  2007;2(9):e825.
For unknown reasons, urinary tract infections (UTIs) are clustered in certain individuals. Here we propose a novel, genetically determined cause of susceptibility to acute pyelonephritis, which is the most severe form of UTI. The IL-8 receptor, CXCR1, was identified as a candidate gene when mIL-8Rh mutant mice developed acute pyelonephritis (APN) with severe tissue damage.
Methods and Findings
We have obtained CXCR1 sequences from two, highly selected APN prone patient groups, and detected three unique mutations and two known polymorphisms with a genotype frequency of 23% and 25% compared to 7% in controls (p<0.001 and p<0.0001, respectively). When reflux was excluded, 54% of the patients had CXCR1 sequence variants. The UTI prone children expressed less CXCR1 protein than the pediatric controls (p<0.0001) and two sequence variants were shown to impair transcription.
The results identify a genetic innate immune deficiency, with a strong link to APN and renal scarring.
PMCID: PMC1950574  PMID: 17786197
13.  ADAMTS13 phenotype in plasma from normal individuals and patients with thrombotic thrombocytopenic purpura 
European Journal of Pediatrics  2006;166(3):249-257.
The activity of ADAMTS13, the von Willebrand factor cleaving protease, is deficient in patients with thrombotic thrombocytopenic purpura (TTP). In the present study, the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting. Normal plasma (n = 20) revealed a single band at 190 kD under reducing conditions using a polyclonal antibody, and a single band at 150 kD under non-reducing conditions using a monoclonal antibody. ADAMTS13 was not detected in the plasma from patients with congenital TTP (n = 5) by either antibody, whereas patients with acquired TTP (n = 2) presented the normal phenotype. Following immunoadsorption of immunoglobulins, the ADAMTS13 band was removed from the plasma of the patients with acquired TTP, but not from that of normal individuals. This indicates that ADAMTS13 is complexed with immunoglobulin in these patients. The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis, impaired cellular secretion, or enhanced degradation in the circulation. This study differentiated between normal and TTP plasma, as well as between congenital and acquired TTP. This method may, therefore, be used as a complement in the diagnosis of TTP.
PMCID: PMC1820762  PMID: 17187257
ADAMTS13; von Willebrand factor; Thrombotic thrombocytopenic purpura; Immunoblotting; Plasma; von Willebrand factor cleaving protease
14.  Interleukin 8 Receptor Deficiency Confers Susceptibility to Acute Experimental Pyelonephritis and May Have a Human Counterpart 
Neutrophils migrate to infected mucosal sites that they protect against invading pathogens. Their interaction with the epithelial barrier is controlled by CXC chemokines and by their receptors. This study examined the change in susceptibility to urinary tract infection (UTI) after deletion of the murine interleukin 8 receptor homologue (mIL-8Rh). Experimental UTIs in control mice stimulated an epithelial chemokine response and increased chemokine receptor expression. Neutrophils migrated through the tissues to the epithelial barrier that they crossed into the lumen, and the mice developed pyuria. In mIL-8Rh knockout (KO) mice, the chemokine response was intact, but the epithelial cells failed to express IL-8R, and neutrophils accumulated in the tissues. The KO mice were unable to clear bacteria from kidneys and bladders and developed bacteremia and symptoms of systemic disease, but control mice were fully resistant to infection. The experimental UTI model demonstrated that IL-8R–dependent mechanisms control the urinary tract defense, and that neutrophils are essential host effector cells. Patients prone to acute pyelonephritis also showed low CXC chemokine receptor 1 expression compared with age-matched controls, suggesting that chemokine receptor expression may also influence the susceptibility to UTIs in humans. The results provide a first molecular clue to disease susceptibility of patients prone to acute pyelonephritis.
PMCID: PMC2193279  PMID: 10993918
urinary tract infection; chemokine receptor; mucosal immunity; lipopolysaccharide; knockout mice
15.  Apoptosis of Renal Cortical Cells in the Hemolytic-Uremic Syndrome: In Vivo and In Vitro Studies 
Infection and Immunity  1998;66(2):636-644.
This study examined apoptotic cell death associated with Shiga-like toxin (Stx)-producing Escherichia coli. Renal cortices from three children with postenteropathic hemolytic-uremic syndrome (HUS) and from mice infected with E. coli O157:H7 and pediatric renal tubular epithelial cells stimulated with Stx and E. coli O157:H7 extracts were examined for apoptotic changes. Apoptotic cells were detected by terminal dUTP nick end labeling of tubuli and glomeruli from HUS patients and from mice inoculated with Stx-2-positive and Stx-negative strains. Apoptosis was more extensive and severe ultramorphological nuclear and cytoplasmic changes were seen in the Stx-2-positive group. Stx caused DNA fragmentation and ultramorphological changes indicating apoptosis in cultured pediatric tubular cells. DNA fragmentation increased when cells were prestimulated with tumor necrosis factor alpha. Polymyxin extracts from Stx-2-positive and Stx-negative strains induced DNA fragmentation, but only extracts from Stx-2-positive strains caused ultramorphological changes and extensive DNA fragmentation. The results indicate that HUS is accompanied by increased apoptosis of kidney cells and that bacterial factors, possibly together with host cytokines in vivo, may activate apoptotic tissue injury.
PMCID: PMC107951  PMID: 9453620

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