Nataliya Lutay

Toll-like receptor 4 promoter polymorphisms: common TLR4 variants may protect against severe urinary tract infection.

Background 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. Conclusions The results suggest that genetic variation in the TLR4 promoter may be an essential, largely overlooked mechanism to influence TLR4 expression and UTI susceptibility.

Inhibition of TIR Domain Signaling by TcpC: MyD88-Dependent and Independent Effects on Escherichia coli Virulence

Toll-like receptor signaling requires functional Toll/interleukin-1 (IL-1) receptor (TIR) domains to activate innate immunity. By producing TIR homologous proteins, microbes inhibit host response induction and improve their own survival. The TIR homologous protein TcpC was recently identified as a virulence factor in uropathogenic Escherichia coli (E. coli), suppressing innate immunity by binding to MyD88. This study examined how the host MyD88 genotype modifies the in vivo effects of TcpC and whether additional, TIR-domain containing proteins might be targeted by TcpC. In wild type mice (wt), TcpC enhanced bacterial virulence, increased acute mortality, bacterial persistence and tissue damage after infection with E. coli CFT073 (TcpC+), compared to a ΔTcpC deletion mutant. These effects were attenuated in Myd88−/− and Tlr4−/− mice. Transcriptomic analysis confirmed that TcpC inhibits MYD88 dependent gene expression in CFT073 infected human uroepithelial cells but in addition the inhibitory effect included targets in the TRIF and IL-6/IL-1 signaling pathways, where MYD88 dependent and independent signaling may converge. The effects of TcpC on bacterial persistence were attenuated in Trif −/− or Il-1β −/− mice and innate immune responses to ΔTcpC were increased, confirming that Trif and Il-1β dependent targets might be involved in vivo, in addition to Myd88. Furthermore, soluble TcpC inhibited Myd88 and Trif dependent TLR signaling in murine macrophages. Our results suggest that TcpC may promote UTI-associated pathology broadly, through inhibition of TIR domain signaling and downstream pathways. Dysregulation of the host response by microbial TcpC thus appears to impair the protective effects of innate immunity, while promoting inflammation and tissue damage.

TLR- and CXCR1-dependent innate immunity: insights into the genetics of urinary tract infections.

The susceptibility to urinary tract infection (UTI) is controlled by the innate immune response and Toll like receptors (TLRs) are the sentinels of this response. If productive, TLR4 signalling may initiate the symptomatic disease process. In the absence of TLR4 signalling the infected host instead develops an asymptomatic carrier state. The activation of mucosal TLR4 is also influenced by the properties of the infecting strain, and pathogens use their virulence factors to trigger ‘pathogen‐specific’ TLR4 responses in the urinary tract but do not respond to the asymptomatic carrier strains in patients with asymptomatic bacteriuria (ABU). The TLR4 dependence has been demonstrated in mice and the relevance of low TLR4 function for protection for human disease was recently confirmed in children with asymptomatic bacteriuria, who expressed less TLR4 than age matched controls. Functional chemokines and functional chemokine receptors are crucial for neutrophil recruitment, and for the neutrophil dependent bacterial clearance. Interleukin (IL)‐8 receptor deficient mice develop acute septic infections and chronic tissue damage, due to aberrant neutrophil function. This mechanism is relevant for human UTI as pyelonephritis prone children express low levels of the human CXCL8 (Il‐8) receptor, CXC chemokine receptor 1 (CXCR1) and often have heterozygous CXCR1 polymorphisms.

Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney Infection

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.

Bacterial control of host gene expression through RNA polymerase II

The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coli develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II–dependent (Pol II–dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coli 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II–dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur.

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.

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.

Mycobacteria bypass mucosal NF-kB signalling to induce an epithelial anti-inflammatory IL-22 and IL-10 response.

The mechanisms by which mycobacteria subvert the inflammatory defence to establish chronic infection remain an unresolved question in the pathogenesis of tuberculosis. Using primary epithelial cells, we have analysed mycobacteria induced epithelial signalling pathways from activation of TLRs to cytokine secretion. Mycobacterium bovis bacilli Calmette-Guerin induced phosphorylation of glycogen synthase kinase (GSK)3 by PI3K–Akt in the signalling pathway downstream of TLR2 and TLR4. Mycobacteria did not supress NF-κB by activating the peroxisome proliferator-activated receptor γ. Instead the pro-inflammatory NF-κB was bypassed by mycobacteria induced GSK3 inhibition that promoted the anti-inflammatory transcription factor CREB. Mycobacterial infection did not thus induce mucosal pro-inflammatory response as measured by TNFα and IFNγ secretion, but led to an anti-inflammatory IL-10 and IL-22 production. Apart from CREB, MAP3Ks p38 and ERK1/2 activated the transcription factor AP-1 leading to IL-6 production. Interestingly, blocking of TLR4 before infection decreased epithelial IL-6 secretion, but increased the CREB-activated IL-10 production. Our data indicate that mycobacteria supress epithelial pro-inflammatory production by supressing NF-κB activation thereby shifting the infection towards an anti-inflammatory state. This balance between the host immune response and the pathogen could determine the outcome of infection.

Survival in amoeba—a major selection pressure on the presence of bacterial copper and zinc resistance determinants? Identification of a “copper pathogenicity island”

The presence of metal resistance determinants in bacteria usually is attributed to geological or anthropogenic metal contamination in different environments or associated with the use of antimicrobial metals in human healthcare or in agriculture. While this is certainly true, we hypothesize that protozoan predation and macrophage killing are also responsible for selection of copper/zinc resistance genes in bacteria. In this review, we outline evidence supporting this hypothesis, as well as highlight the correlation between metal resistance and pathogenicity in bacteria. In addition, we introduce and characterize the “copper pathogenicity island” identified in Escherichia coli and Salmonella strains isolated from copper- and zinc-fed Danish pigs.

The dynamics of platelet activation during the progression of streptococcal sepsis

Platelets contribute to inflammation however, the role of platelet activation during the pathophysiological response to invasive bacterial infection and sepsis is not clear. Herein, we have investigated platelet activation in a mouse model of invasive Streptococcus pyogenes infection at 5, 12, and 18 hours post infection and correlated this to parameters of infection. The platelet population in ex-vivo blood samples showed no increased integrin activation or surface presentation of CD62P, however platelet-neutrophil complex formation and plasma levels of CD62P were increased during bacterial dissemination and the progression of sepsis, indicating that platelet activation had occurred in vivo. Platelet-neutrophil complex formation was the most discriminatory marker of platelet activation. Platelet-neutrophil complexes were increased above baseline levels during early sepsis but decreased to significantly lower levels than baseline during late sepsis. The removal of these complexes from the circulation coincided with a significant increase in organ damage and the accumulation of platelets in the liver sinusoids, suggesting that platelet activation in the circulation precedes accumulation of platelets in damaged organs. The results demonstrate that monitoring platelet activation using complementary methods may provide prognostic information during the pathogenesis of invasive S. pyogenes infection.