Long-term prevention of bladder cancer progression by alpha1-oleate alone or in combination with chemotherapy.

Bladder cancer is common and one of the most costly cancer forms, due to a lack of curative therapies. Recently, clinical safety and efficacy of the alpha1-oleate complex was demonstrated in a placebo-controlled study of nonmuscle invasive bladder cancer. Our study investigated if long-term therapeutic efficacy is improved by repeated treatment cycles and by combining alpha1-oleate with low-dose chemotherapy. Rapidly growing bladder tumors were treated by intravesical instillation of alpha1-oleate, Epirubicin or Mitomycin C alone or in combination. One treatment cycle arrested tumor growth, with a protective effect lasting at least 4 weeks in mice receiving 8.5 mM of alpha1-oleate alone or 1.7 mM of alpha-oleate combined with Epirubicin or Mitomycin C. Repeated treatment cycles extended protection, defined by a lack of bladder pathology and a virtual absence of bladder cancer-specific gene expression. Synergy with Epirubicin was detected at the lower alpha1-oleate concentration and in vitro, alpha1-oleate was shown to enhance the uptake and nuclear translocation of Epirubicin, by tumor cells. Effects at the chromatin level affecting cell proliferation were further suggested by reduced BrdU incorporation. In addition, alpha1-oleate triggered DNA fragmentation, defined by the TUNEL assay. The results suggest that bladder cancer development may be prevented long-term in the murine model, by alpha1-oleate alone or in combination with low-dose Epirubicin. In addition, the combination of alpha1-oleate and Epirubicin reduced the size of established tumors. Exploring these potent preventive and therapeutic effects will be of immediate interest in patients with bladder cancer.

The humoral pattern recognition molecule PTX3 is a key component of innate immunity against urinary tract infection.

Immunity in the urinary tract has distinct and poorly understood pathophysiological characteristics and urinary tract infections (UTIs) are important causes of morbidity and mortality. We investigated the role of the soluble pattern recognition molecule pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity, in UTIs. PTX3-deficient mice showed defective control of UTIs and exacerbated inflammation. Expression of PTX3 was induced in uroepithelial cells by uropathogenic Escherichia coli (UPEC) in a Toll-like receptor 4 (TLR4)- and MyD88-dependent manner. PTX3 enhanced UPEC phagocytosis and phagosome maturation by neutrophils. PTX3 was detected in urine of UTI patients and amounts correlated with disease severity. In cohorts of UTI-prone patients, PTX3 gene polymorphisms correlated with susceptibility to acute pyelonephritis and cystitis. These results suggest that PTX3 is an essential component of innate resistance against UTIs. Thus, the cellular and humoral arms of innate immunity exert complementary functions in mediating resistance against UTIs.

Peptide-Oleate Complexes Create Novel Membrane-Bound Compartments.

A challenging question in evolutionary theory is the origin of cell division and plausible molecular mechanisms involved. Here, we made the surprising observation that complexes formed by short alpha-helical peptides and oleic acid can create multiple membrane-enclosed spaces from a single lipid vesicle. The findings suggest that such complexes may contain the molecular information necessary to initiate and sustain this process. Based on these observations, we propose a new molecular model to understand protocell division.

Fimbriae reprogram host gene expression - Divergent effects of P and type 1 fimbriae.

Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-ß and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors.

Bladder cancer therapy without toxicity-A dose-escalation study of alpha1-oleate.

Potent chemotherapeutic agents are required to counteract the aggressive behavior of cancer cells and patients often experience severe side effects, due to tissue toxicity. Our study addresses if a better balance between efficacy and toxicity can be attained using the tumoricidal complex alpha1-oleate, formed by a synthetic, alpha-helical peptide comprising the N-terminal 39 amino acids of alpha-lactalbumin and the fatty acid oleic acid. Bladder cancer was established, by intravesical instillation of MB49 cells on day 0 and the treatment group received five instillations of alpha1-oleate (1.7-17 mM) on days 3 to 11. A dose-dependent reduction in tumor size, bladder size and bladder weight was recorded in the alpha1-oleate treated group, compared to sham-treated mice. Tumor markers Ki-67, Cyclin D1 and VEGF were inhibited in a dose-dependent manner, as was the expression of cancer-related genes. Remarkably, toxicity for healthy tissue was not detected in alpha1-oleate-treated, tumor-bearing mice or healthy mice or rabbits, challenged with increasing doses of the active complex. The results define a dose-dependent therapeutic effect of alpha1-oleate in a murine bladder cancer model.

Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets.

Tissue damage is usually regarded as a necessary price to pay for successful elimination of pathogens by the innate immune defense. Yet, it is possible to distinguish protective from destructive effects of innate immune activation and selectively attenuate molecular nodes that create pathology. Here, we identify acute cystitis as an Interleukin-1 beta (IL-1ß)-driven, hyper-inflammatory condition of the infected urinary bladder and IL-1 receptor blockade as a novel therapeutic strategy. Disease severity was controlled by the mechanism of IL-1ß processing and mice with intact inflammasome function developed a moderate, self-limiting form of cystitis. The most severe form of acute cystitis was detected in mice lacking the inflammasome constituents ASC or NLRP-3. IL-1ß processing was hyperactive in these mice, due to a new, non-canonical mechanism involving the matrix metalloproteinase 7- (MMP-7). ASC and NLRP-3 served as transcriptional repressors of MMP7 and as a result, Mmp7 was markedly overexpressed in the bladder epithelium of Asc-/- and Nlrp3-/- mice. The resulting IL-1ß hyper-activation loop included a large number of IL-1ß-dependent pro-inflammatory genes and the IL-1 receptor antagonist Anakinra inhibited their expression and rescued susceptible Asc-/- mice from bladder pathology. An MMP inhibitor had a similar therapeutic effect. Finally, elevated levels of IL-1ß and MMP-7 were detected in patients with acute cystitis, suggesting a potential role as biomarkers and immunotherapeutic targets. The results reproduce important aspects of human acute cystitis in the murine model and provide a comprehensive molecular framework for the pathogenesis and immunotherapy of acute cystitis, one of the most common infections in man. TRIAL REGISTRATION: The clinical studies were approved by the Human Ethics Committee at Lund University (approval numbers LU106-02, LU236-99 and Clinical Trial Registration RTP-A2003, International Committee of Medical Journal Editors, www.clinicaltrials.gov).

HAMLET - A protein-lipid complex with broad tumoricidal activity.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.

Innate immunity and genetic determinants of urinary tract infection susceptibility.

PURPOSE OF REVIEW: Urinary tract infections (UTIs) are common, dangerous and interesting. Susceptible individuals experience multiple, often clustered episodes, and in a subset of patients, infections progress to acute pyelonephritis (APN), sometimes accompanied by uro-sepsis. Others develop asymptomatic bacteriuria (ABU). Here, we review the molecular basis for these differences, with the intention to distinguish exaggerated host responses that drive disease from attenuated responses that favour protection and to highlight the genetic basis for these extremes, based on knock-out mice and clinical studies. RECENT FINDINGS: The susceptibility to UTI is controlled by specific innate immune signalling and by promoter polymorphisms and transcription factors that modulate the expression of genes controlling these pathways. Gene deletions that disturb innate immune activation either favour asymptomatic bacteriuria or create acute morbidity and disease. Promoter polymorphisms and transcription factor variants affecting those genes are associated with susceptibility in UTI-prone patients. SUMMARY: It is time to start using genetics in UTI-prone patients, to improve diagnosis and to assess the risk for chronic sequels such as renal malfunction, hypertension, spontaneous abortions, dialysis and transplantation. Furthermore, the majority of UTI patients do not need follow-up, but for lack of molecular markers, they are unnecessarily investigated.

Polyethyleneimine is a potent systemic adjuvant for glycoprotein antigens.

Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens.

Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells).

BACKGROUND: Most colon cancers start with dysregulated Wnt/ß-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. OBJECTIVE: To investigate if HAMLET can be used for colon cancer treatment and prevention. Apc(Min)(/+) mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. METHOD: HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/ß-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. RESULTS: Peroral HAMLET administration reduced tumour progression and mortality in Apc(Min)(/+) mice. HAMLET accumulated specifically in tumour tissue, reduced ß-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered ß-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling ß-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. CONCLUSIONS: These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death.

Lipids as tumoricidal components of human α-lactalbumin made lethal to tumor cells (HAMLET): unique and shared effects on signaling and death.

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 µm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 µm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

Rare emergence of symptoms during long-term asymptomatic Escherichia coli 83972 carriage without an altered virulence factor repertoire.

PURPOSE: Asymptomatic bacteriuria established by intravesical inoculation of Escherichia coli 83972 is protective in patients with recurrent urinary tract infections. In this randomized, controlled crossover study a total of 3 symptomatic urinary tract infection episodes developed in 2 patients while they carried E. coli 83972. We examined whether virulence reacquisition by symptom isolates may account for the switch from asymptomatic bacteriuria to symptomatic urinary tract infection. MATERIALS AND METHODS: We used E. coli 83972 re-isolates from 2 patients in a prospective study and from another 2 in whom symptoms developed after study completion. We phylogenetically classified the re-isolates, and identified the genomic restriction patterns and gene expression profiles as well as virulence gene structure and phenotypes. In vivo virulence was examined in the murine urinary tract infection model. RESULTS: The fim, pap, foc, hlyA, fyuA, iuc, iroN, kpsMT K5 and malX genotypes of the symptomatic re-isolates remained unchanged. Bacterial gene expression profiles of flagellated symptomatic re-isolates were unique to each host, providing no evidence of common deregulation. Symptomatic isolates did not differ in virulence from the wild-type strain, as defined in the murine urinary tract infection model by persistence, symptoms or innate immune activation. CONCLUSIONS: The switch from asymptomatic E. coli 83972 carriage to symptomatic urinary tract infection was not explained by reversion to a functional virulence gene repertoire.

Similar immune responses to alpha1-oleate and Bacillus Calmette-Guérin treatment in patients with bladder cancer.

BACKGROUND: The molecular content of urine is defined by filtration in the kidneys and by local release from tissues lining the urinary tract. Pathological processes and different therapies change the molecular composition of urine and a variety of markers have been analyzed in patients with bladder cancer. The response to BCG immunotherapy and chemotherapy has been extensively studied and elevated urine concentrations of IL-1RA, IFN-α, IFN-γ TNF-α, and IL-17 have been associated with improved outcome. METHODS: In this study, the host response to intravesical alpha 1-oleate treatment was characterized in patients with non-muscle invasive bladder cancer by proteomic and transcriptomic analysis. RESULTS: Proteomic profiling detected a significant increase in multiple cytokines in the treatment group compared to placebo. The innate immune response was strongly activated, including IL-1RA and pro-inflammatory cytokines in the IL-1 family (IL-1α, IL-1ß, IL-33), chemokines (MIP-1α, IL-8), and interferons (IFN-α2, IFN-γ). Adaptive immune mediators included IL-12, Granzyme B, CD40, PD-L1, and IL-17D, suggesting broad effects of alpha 1-oleate treatment on the tumor tissues. CONCLUSIONS: The cytokine response profile in alpha 1-oleate treated patients was similar to that reported in BCG treated patients, suggesting a significant overlap. A reduction in protein levels at the end of treatment coincided with inhibition of cancer-related gene expression in tissue biopsies, consistent with a positive treatment effect. Thus, in addition to killing tumor cells and inducing cell detachment, alpha 1-oleate is shown to activate a broad immune response with a protective potential.

BAMLET administration via drinking water inhibits intestinal tumor development and promotes long-term health.

Though new targeted therapies for colorectal cancer, which progresses from local intestinal tumors to metastatic disease, are being developed, tumor specificity remains an important problem, and side effects a major concern. Here, we show that the protein-fatty acid complex BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) can act as a peroral treatment for colorectal cancer. ApcMin/+ mice, which carry mutations relevant to hereditary and sporadic human colorectal cancer, that received BAMLET in the drinking water showed long-term protection against tumor development and decreased expression of tumor growth-, migration-, metastasis- and angiogenesis-related genes. BAMLET treatment via drinking water inhibited the Wnt/ß-catenin and PD-1 signaling pathways and prolonged survival without evidence of toxicity. Systemic disease in the lungs, livers, spleens, and kidneys, which accompanied tumor progression, was inhibited by BAMLET treatment. The metabolic response to BAMLET included carbohydrate and lipid metabolism, which were inhibited in tumor prone ApcMin/+ mice and weakly regulated in C57BL/6 mice, suggesting potential health benefits of peroral BAMLET administration in addition to the potent antitumor effects. Together, these findings suggest that BAMLET administration in the drinking water maintains antitumor pressure by removing emergent cancer cells and reprogramming gene expression in intestinal and extra-intestinal tissues.

Electrostatic interactions play an essential role in the binding of oleic acid with α-lactalbumin in the HAMLET-like complex: a study using charge-specific chemical modifications.

Human α-lactalbumin made lethal to tumor cells (HAMLET) and its analogs are partially unfolded protein-oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge-specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively-charged lysine residues to negatively-charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild-type α-lactalbumin-oleic acid complex. With the addition of OA, the wild-type and guanidinated α-lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α-lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively-charged basic groups on α-lactalbumin and the negatively-charged carboxylate groups on OA molecules play an essential role in the binding of OA to α-lactalbumin and that these interactions appear to be as important as hydrophobic interactions.

Urinary tract infections in children: microbial virulence versus host susceptibility.

Urinary tract infections (UTI) are common, dangerous and interesting. This review includes a general background on UTIs and molecular mechanisms of pathogenesis. In addition, we discuss UTI susceptibility and especially the effect of genetic variation on innate immunity. The symptoms of acute pyelonephritis are caused by the innate immune response and inflammation in the urinary tract decreases renal tubular function and may give rise to renal scarring, especially in childhood. The disease severity is explained by pathogens and their virulence factors triggering signaling through Toll-like receptors (TLRs), interferon regulatory factors (IRFs) and type 1 interferons, and the activation of a host response mediating disease or pathology or clearance of infection. In children with asymptomatic bacteriuria (ABU), in contrast, bacteria persist without causing symptoms or pathology. ABU strains mostly lack virulence factors, and the lack of symptoms has largely been attributed to their lack of virulence. Recently, rapid progress has been made in the understanding of host susceptibility mechanisms. For example, genetic alterations that reduce TLR4 function are associated with ABU while polymorphisms reducing IRF3 or CXCR1 expression are associated with acute pyelonephritis and an increased risk for renal scarring. Understanding bacterial virulence and host resistance promises new tools to improve the diagnostic accuracy in children with UTI. By combining information on bacterial virulence and the host response, it should be possible to start individualizing diagnosis and therapy. Finally, we propose that the prediction of future disease risk and decisions on prophylaxis and invasive diagnostic procedures might be improved by genetic analysis.

Therapeutic Effects of IL-1RA against Acute Bacterial Infections, including Antibiotic-Resistant Strains.

Innate immunity is essential for the anti-microbial defense, but excessive immune activation may cause severe disease. In this study, immunotherapy was shown to prevent excessive innate immune activation and restore the anti-bacterial defense. E. coli-infected Asc-/- mice develop severe acute cystitis, defined by IL-1 hyper-activation, high bacterial counts, and extensive tissue pathology. Here, the interleukin-1 receptor antagonist (IL-1RA), which inhibits IL-1 hyper-activation in acute cystitis, was identified as a more potent inhibitor of inflammation and NK1R- and substance P-dependent pain than cefotaxime. Furthermore, IL-1RA treatment inhibited the excessive innate immune activation in the kidneys of infected Irf3-/- mice and restored tissue integrity. Unexpectedly, IL-1RA also accelerated bacterial clearance from infected bladders and kidneys, including antibiotic-resistant E. coli, where cefotaxime treatment was inefficient. The results suggest that by targeting the IL-1 response, control of the innate immune response to infection may be regained, with highly favorable treatment outcomes, including infections caused by antibiotic-resistant strains.

Comparison of asymptomatic bacteriuria Escherichia coli isolates from healthy individuals versus those from hospital patients shows that long-term bladder colonization selects for attenuated virulence phenotypes.

Asymptomatic bacteriuria (ABU) is a condition where bacteria stably colonize the urinary tract, in a manner closely resembling commensalism at other mucosal sites. The patients carry >10(5) CFU/ml for extended periods of time and rarely develop symptoms. Contrasting the properties of ABU strains to those of uropathogenic isolates causing symptomatic infection is therefore highly relevant to understand mechanisms of bacterial adaptation. The prototype ABU strain Escherichia coli 83972 has a smaller genome than uropathogenic E. coli (UPEC) strains with deletions or point mutations in several virulence genes, suggesting that ABU strains undergo a programmed reductive evolution within human hosts. This study addressed if these observations can be generalized. Strains causing ABU in outpatients or hospitalized patients after catheterization or other invasive procedures were compared to commensal E. coli isolates from the intestinal flora of healthy individuals. Notably, clonal complex 73 (CC73) was a prominent phylogenetic lineage dominated by ABU isolates. ABU isolates from outpatients and hospitalized patients had a similar overall virulence gene repertoire, which distinguished them from many commensals, but typical UPEC virulence genes were less frequently attenuated in hospital strains than in outpatient strains or commensals. The decreased virulence potential of outpatient ABU isolates relative to that of ABU strains from hospitalized patients supports the hypothesis that loss of expression or decay of virulence genes facilitates long-term carriage and adaptation to host environments.

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-1ß (-/-) mice and innate immune responses to ΔTcpC were increased, confirming that Trif and Il-1ß 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.

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/IFNß-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.