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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.
Citation: Fischer H, Lutay N, Ragnarsdóttir B, Yadav M, Jönsson K, Urbano A, et al. (2010) Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney Infection. PLoS Pathog 6(9): e1001109. doi:10.1371/journal.ppat.1001109
Editor: Frederick M. Ausubel, Massachusetts General Hospital and Harvard Medical School, United States of America
Received: March 4, 2010; Accepted: August 17, 2010; Published: September 23, 2010
Copyright: © 2010 Fischer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The studies were supported by grants from the Swedish Medical Research Council (http://www.vr.se/), the Crafoord (http://www.crafoord.se/), Wallenberg (http://www.wallenberg.com/), Lundberg (http://web.lundbergsstiftelserna.se/), Sweden-Japan (http://www.swejap.a.se/), Swedish Institute (http://www.si.se/) and Osterlund Foundations and the Royal Physiographic Society (http://www.fysiografen.se/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Despite significant advances in the understanding of genetic variation, common infections are often regarded as too complex for genetic analysis. While single gene defects have a major impact on host susceptibility to classic infections like malaria [1], the extent to which susceptibility to diarrhea, respiratory tract and urinary tract infection (UTI) is genetically controlled remains unclear. Critical to the understanding of host resistance and genetic control is the mucosal route of these infections and the molecular interactions through which mucosal tissues are perturbed. UTI serve as a particularly useful model to identify genetic variants contributing to host susceptibility, as innate immunity controls the antimicrobial defense and molecular mechanisms of host parasite interaction are understood in great detail [2], [3]. The disease response to uropathogenic Escherichia coli is initiated through fimbriae-mediated adherence, and the expression of P fimbriae distinguishes the pathogenic strains from non-virulent bacteria, which colonize the same mucosal sites.
TLRs control the survival of complex organisms by balancing protective against destructive forces of innate immunity. During infection, each TLR recognizes a relatively small number of ligands, including conserved microbial patterns (PAMPs) [4]. The horseshoe-shaped, leucine-rich, extracellular TLR domain and its co-receptors are involved in recognition of proteins, as well as lipids, carbohydrates and nucleic acids [5], [6], [7]. At mucosal sites, where the bulk of microbial challenge occurs, PAMP recognition is non-functional, however, and does not explain how mucosal TLRs distinguish pathogenic microbes from members of the normal flora [8]. Pathogen-specific TLR responses to mucosal pathogens require receptors that exclusively engage virulence ligands and signaling pathways that activate a pathogen-specific defense [8]. For example, uropathogenic E. coli adhere to mucosa via glycosphingolipid receptors for P fimbriae, thereby activating a TLR4-dependent but LPS/CD14-independent innate immune response in epithelial cells [9].
Signaling through cell surface sphingolipids involves ceramide, the membrane anchor and a ubiquitous component of cell membranes [10], [11]. The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms, clustering receptor molecules and facilitating signal transduction following receptor stimulation [12]. Endogenous SMases, activated by many infectious agents, cleave ceramide from the extracellular choline-rich domain of sphingomyelin [13], [14], [15], [16], [17] and activate the "ceramide-signaling pathway", which is conserved from yeast to humans [18]. In addition, pathogens that utilize the extracellular domain of glycosphingolipids as receptors may release ceramide after bacterial binding, as first described for P-fimbriated, uropathogenic E. coli [9], [17], [19]. Ceramide activates a TLR4-dependent innate immune response [17], similar to infection-mediated activation, and we have proposed that ceramide acts as a signaling intermediate between the pathogen-specific receptors and TLR4 [9], [17], [19]. The molecular mechanisms in this important signal transduction need to be identified, however.
UTIs affect >150 million adults each year and about 5% of children <12 years of age. Severe kidney infections like acute pyelonephritis (APN) are accompanied by life-threatening urosepsis in about 30% of adults. Children may develop renal scars, which are associated with long-term morbidity including hypertension, complications of pregnancy, and renal failure if scarring is extensive. Despite the urgent need, no tools exist at present to identify children at risk of developing recurrent acute pyelonephritis and renal scarring. Host resistance to UTI is controlled by the innate immune system, through Toll-like receptor (TLR) activation [8], [9], [20]. Previous studies have shown that TLR4-deficient mice develop asymptomatic carriage rather than severe disease [8], [20], [21], suggesting that disturbances in TLR4 signaling may alter the innate immune dependent host defense [22].
This study examined TLR4 activation by ceramide and P fimbriated E. coli and characterized this signaling pathway. We propose that ceramide interacts directly with TLR4, activates TRAM phosphorylation followed by nuclear translocation of IRF3. Furthermore, we show that IRF3 dependent innate immunity is essential for the host defense, as Irf3 knockout mice develop severe kidney infection. Finally, we show that IRF3 promoter polymorphisms are more common in APN prone patients than in those who become asymptomatic bacterial carriers. We propose that this pathway offers a model of how TLR4 may distinguish pathogens from commensals at the mucosal level, through modification of pathogen recognition receptors, adaptors and transcription factors.
Increased susceptibility to acute pyelonephritis in Irf3−/− mice
In a genetic screen of innate immune effector genes downstream of TLR4, we identified IRF3 as a major determinant of host susceptibility. Irf3−/− and Irf3+/+ mice were infected via the urinary tract mucosal route with the uropathogenic E. coli strain CFT073 [23]. The Irf3−/− mice developed more severe disease than wild type (wt) Irf3+/+ mice. Acute mortality was higher (50% after 24 hours) and bacterial clearance was significantly impaired, with higher bacterial counts in urine, kidneys and bladders (Figure 1A, p<0.001). Abscess formation was also more extensive in Irf3−/− than in wt mice (day 7 post-infection, Figure 1B–C, p<0.001). In Irf3−/− mice, abscesses were diffuse, destroying large tissue areas while in wt mice abscesses were morphologically distinct from surrounding healthy tissue (Figure 1B).
Uropathogenic E. coli CFT073 established acute pyelonephritis more efficiently in Irf3−/− (n = 11) compared to Irf3+/+ (n = 10) mice. Panel A shows increased mortality (*** p<0.001), bacterial numbers in urine, kidneys and bladders (** p<0.01, *** p<0.001) and neutrophil influx (*** p<0.001). Geometric means ± SEMs. Panels B–D show destruction of renal tissue in Irf3−/− mice. Sections were stained with hematoxylin-eosin or specific antibodies (neutrophils = green, NIMP-R14, PapG adhesin = red, synthetic PapG peptide antibodies and Nuclear Hoechst staining = blue). Scale bar = 200 mm. Abscesses are indicated by arrows. Panel E shows that non-virulent E. coli 83972 are cleared with similar efficiency by wt (n = 8) and Irf3−/− (n = 8) mice. In contrast, a P-fimbriated transformant E. coli 83972pap, triggers IRF3-dependent disease. Geometric means ± SEMs (** p<0.01).
Renal abscess formation is caused, in part, by an imbalance between neutrophil recruitment and exit from the tissues [24], [25]. The kinetics of early neutrophil recruitment did not differ between wild type and Irf3−/− mice (Figure 1A), but later, neutrophil recruitment subsided in wt mice but remained elevated in Irf3−/− mice. In tissue sections from Irf3−/− mice, neutrophils were detected throughout the abscesses and P-fimbriated bacteria were interspersed among the neutrophils, as shown by PapG adhesin-specific antibody detection (Figure 1D, for negative control, see Figure S1). Wt mice, in contrast, had discrete, neutrophil aggregates with fewer bacteria. The results suggest that Irf3 is essential for a functioning innate immune defense against UTI, to maintain tissue integrity and to clear mucosal E. coli infection.
To examine if the IRF3-dependent immune response discriminates uropathogenic E. coli from non-pathogenic bacteria, we inoculated wt and Irf3−/− mice with the prototypical asymptomatic bacteriuria strain E. coli 83972, which lacks functional UTI-associated virulence factors, including P fimbriae [26], [27], [28], [29]. Both wt and Irf3−/− mice cleared infection rapidly, with no difference in bacterial counts (Figure 1E) and no significant neutrophil recruitment (data not shown). As P fimbriae are essential virulence factors, present in up to 100% of E. coli strains causing urosepsis [30], [31], we subsequently examined if P fimbriae activate the IRF3 pathway. The asymptomatic carrier strain E. coli 83972 was transformed with a chromosomal copy of the pap gene cluster. We compared disease severity and bacterial counts between wt and Irf3−/− mice infected with E. coli 83972pap. The Irf3−/− mice developed acute, symptomatic disease with sepsis and had dramatically increased bacterial numbers in bladders, kidneys and spleens (Figure 1E, p<0.05), compared to wt mice, which were resistant to infection with E. coli 83972pap.
The results show that the IRF3-dependent response distinguishes pathogenic E. coli from non-pathogenic strains and suggest that the expression of a single virulence factor like P fimbriae enables the host to recognize a potential pathogen and to activate this response.
Ceramide/TLR4 interactions and TRAM phosphorylation
P fimbriae bind to glycosphingolipid receptors and trigger ceramide release [9]. To investigate the mechanism of pathogen-specific TLR4/IRF3 signaling activation, we examined if ceramide and TLR4 interact after ceramide release from membrane glycosphingolipids. We treated A498 kidney epithelial cells with sphingomyelinase (SMase) for one hour, to release ceramide (r-ceramide) from the extracellular phosphocholine domain of sphingomyelin [32] (Figure 2A–D). We labeled TLR4 and native ceramide with specific primary antibodies followed by Alexa fluor-488 (donor) and Alexa fluor-568 (acceptor)-labeled secondary antibodies, respectively. In unstimulated cells (no SMase treatment), where ceramide remains bound to sphingomyelin, we detected no FRET signal. After SMase treatment, we recorded a significant FRET signal (Figure 2A–D, 50% FRET-positive cells compared to 8% for unstimulated cells, p<0.05), with most of the FRET-positive regions localized in the plasma membrane. LPS and soluble CD14 (sCD14) stimulation, in contrast, did not stimulate a FRET signal above background (p>0.05, compared to unstimulated cells). sCD14 was used, as the uroepithelial cells lack membrane-bound CD14 and respond poorly to LPS [33]. These results suggest that ceramide interacts with TLR4 after release from membrane glycosphingolipids.
TLR4 and native ceramide were labeled with specific primary antibodies, followed by Alexa fluor-488 (donor) and Alexa fluor-568 (acceptor)-labeled secondary antibodies, respectively. Acceptor bleaching was quantified by confocal microscopy, comparing target membrane regions (inset) and cytoplasmic control areas. Panel A shows membrane and cytoplasmic staining for TLR4 (green) and ceramide (red). Panel B shows recording of donor and acceptor channels, ten times before and after acceptor bleaching (arrow). The intensity at time 0 was set to 100%. Panel C compared cells exposed to LPS+sCD14 (10+1µg/ml) or r-ceramide (SMase, 1 U/ml, 1 h). Panel D shows an increase in membrane FRET signals after ceramide release, but not after LPS+sCD14 treatment. Panel E shows siRNA silencing (black, 72 h transfection) of TLR4, TRAM or MyD88 and resulting inhibition of IL-8 responses in A549 cells, stimulated with SMase (1 U/ml) or LPS+sCD14 (10+1µg/ml). Cytokine responses (Medians±SEMs ≥3 experiments) were compared to irrelevant siRNA-transfected cells (grey, * p<0.05, **p<0.01, ***p<0.001) which gave significant IL-8 response compared to background level (¤p<0.05, ¤¤p<0.01, ¤¤¤p<0.001). Panel F shows TRAM phosphorylation (TRAM-P) after r-ceramide, C6 ceramide or LPS+sCD14 exposure (primary polyclonal rabbit-TRAM-P antibodies and secondary anti-rabbit-Alexa fluor-568-labeled antibodies), for a broader view see Figure S3. Panel G shows TRAM-P fluorescence intensity quantified with LSM 510 software (mean±SEM, n = 30 cells/treatment, * p<0.05, *** p<0.001). (H) TRAM-P expression in A549 cells stimulated for 45 min with the indicated agonists compared to total TRAM levels (Western blot).
To examine the ceramide-induced TLR4 signaling pathway, we used RNA interference to suppress specific genes (Figure 2E, siRNA used for transfection are listed in Table S1 in Supporting Information S1; for knockdown efficiency compared to control cells transfected with irrelevant siRNA, see Figure S2). First, suppression of TLR4 expression abrogated the innate immune response to r-ceramide (p<0.001), confirming that this pathway is TLR4 dependent. Secondly, TRAM siRNA inhibited the responses to r-ceramide (p<0.05 compared to the siRNA control). MyD88-specific siRNA did not alter the ceramide response (p>0.05 compared to the siRNA control) but did reduce the response to LPS+sCD14 (p<0.05), as did TLR4- and TRAM-specific siRNAs.
To further investigate ceramide-induced TLR4 signaling, TRAM phosphorylation (TRAM-P) was quantified by confocal microscopy, using polyclonal phospho-specific anti-TRAM antibodies (Figure 2F–G and Figure S3). We detected an increase in TRAM-P staining in cells exposed to r-ceramide or exogenous, water-soluble C6 ceramide; staining had a granular appearance and was most intense in the perinuclear area. By Western blot analysis (Figure 2H), a band corresponding to TRAM-P was increased in cells exposed to C6 and r-ceramide compared to unstimulated cells but total TRAM levels were not altered. LPS+sCD14 triggered weaker TRAM phosphorylation, as shown by confocal microscopy (p<0.001 compared to r-ceramide) and by Western blot. The results indicate that ceramide triggers TRAM phosphorylation more efficiently than LPS+sCD14. As TRAM phosphorylation was virtually absent in unstimulated cells, this pathway may need to be activated by exogenous or endogenous stimuli.
Kinase phosphorylation downstream of ceramide/TLR4
To define signaling downstream of ceramide/TLR4 and TRAM, we examined kinase phosphorylation, using phosphoarrays specific for 46 protein kinases and substrates (Figure 3A). Ceramide release stimulated the phosphorylation of twelve protein kinases: p27T198, eNOS, CREB, Fyn (all 2.3-fold), Hck, PLCγ1, Jun (all 2.1-fold), Pyk2 (2-fold), ERK1/2 and Src (1.9-fold), RSK1/2/3 (1.8-fold), p27T157 (1.7-fold), and p53 (1.6-fold). Antibacterial effectors included eNOS, which regulates nitric oxide and related antibacterial effector functions [34] and Hck, a Src-family tyrosine kinase associated with secretory lysosomes in neutrophils and phagosome-lysosome fusion [35]. A number of the significantly phosphorylated proteins activate IRF3- and AP1-dependent transcription. PLCγ1 catalyzes the formation of inositol 1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate, leading to PKC activation and CREB (cAMP response element binding) phosphorylation [36], [37]. CREB is then phosphorylated and binds to CBP (CREB-binding protein), which preferentially associates with phosphorylated IRF3 [38], [39], leading to IRF3. Fyn is a Src family tyrosine kinase implicated in the activation of PKA, a protein kinase involved in CREB phosphorylation [40]. Jun in combination with Fos bind to and are a part of the AP-1 transcription factor complex [41], which induces the transcription of proinflammatory cytokines. Pyk2 activation is highly correlated with the stimulation of c-Jun N-terminal kinase (JNK). Identified phosphorylation targets also included ERKs (ERK1/2, extracellular signal-regulated kinases) which activate downstream protein kinases and transcription factors, including IRF3 and AP-1 [42].
Panel A shows a phosphoproteomic heat map of A549 cells stimulated for 60 min with r-ceramide (SMase, 1 U/ml) or LPS+sCD14 (10+1 µg/ml) compared to untreated cells. Fold changes in protein phosphorylation levels were determined, using the Human Phospho-Kinase Array Kit. Panels B and C show gene expression heat maps of human epithelial cell RNAs. A549 or A498 cells were stimulated for 1 h or 3 h with r-ceramide or LPS+sCD14. Panels D and E show CREB phosphorylation (CREB-P) activated by r-ceramide but not by LPS+sCD14 (rabbit anti-human CREB-P primary antibodies). Fos-P activated by r-ceramide but not by LPS+sCD14 (rabbit anti-human Fos-P primary antibodies). JNK phosphorylation (JNK-P) by r-ceramide and LPS+sCD14 (rabbit anti-human JNK-P primary antibodies). Fluorescence intensities were quantified with LSM 510 software. Mean ± SEM, n = 60 cells/treatment, * p<0.05, *** p<0.001, compared to LPS+sCD14 or ¤ p<0.05, ¤¤¤ p<0.001 compared to unstimulated cells.
CREB phosphorylation in r-ceramide-activated cells was confirmed by confocal microscopy (Figure 3D, E, p<0.001 compared to control), but was not detected in LPS-stimulated cells. We obtained similar results using antibodies specific for phosphorylated Fos (Figure 3D, E). JNK phosphorylation, in contrast, was similar after r-ceramide and LPS+sCD14 stimulation (Figure 3D, E), suggesting that JNK signaling was not ceramide-specific (p<0.001 compared to the control). The results suggest that ceramide-induced TLR4 signaling causes rapid phosphorylation/transcription of proteins involved in IRF3 and AP-1 transcription, including CREB, Fyn, PLCγ, MAP kinases, ERK1/2 and Fos/Jun (Figure S4). LPS+sCD14, in contrast, caused a weaker phosphorylation response, comprising p27T198 (2-fold), eNOS (1.8-fold), PLCγ1 (1.7-fold), Pyk2 (1.7-fold), and Jun (1.6-fold), but not the remaining targets that were phosphorylated in response to r-ceramide (Figure 3A).
Transcriptional activation in response to ceramide
Innate immune activation in response to ceramide was further examined by TLR SuperArrays and compared to LPS+sCD14 (Figure 3B, Figure S5). After one hour, five genes in A549 cells had responded to r-ceramide: Fos (6.5-fold) and Jun (2.1-fold), IL-8 (4.4-fold), IL-6 (2.9-fold) and IL-1α (2-fold). The response showed a similar, restricted repertoire in A498 carcinoma cells after three hours (Figure 3C). r-Ceramide upregulated TRAM, Fos and Jun transcription (10.2-, 2.2- and 2.4-fold, respectively). Ceramide activated IL-6 transcription (6.2-fold), MAP3K1 and MAP2K3 (5.9- and 2.5-fold), as well as IL-8 and CSF2 transcription levels (about 3-fold). In contrast, LPS+sCD14 did not significantly stimulate Fos (1.5-fold), Jun (1.2-fold) or IL-1α after one hour. After three hours, only IL-8 transcription was higher in response to LPS+sCD14 than to r-ceramide. The transcriptional profile confirmed the difference between ceramide and LPS+sCD14 activated cells, consistent with a different transcription factor usage.
IRF3 translocation to the nucleus in response to ceramide/TLR4
IRF3 is an interferon regulatory transcription factor and following TLR4 activation, phosphorylated IRF3 homodimers translocate from the cytosol to the nucleus [43], [44], [45], [46]. By confocal microscopy (Figure 4A, C) we observed that r-ceramide triggered IRF3 translocation to the nucleus (p<0.001 compared to unstimulated cells, 90 min). We confirmed the results in a human bladder epithelial cell line (J82, Figure S6A) in which ceramide release caused rapid IRF3 translocation. In cells exposed to LPS+sCD14, the nuclear IRF3 translocation was weak (p>0.05 compared to control) and fewer dimers were formed after exposure to LPS+sCD14. In the bladder epithelial cells, the IRF3 response to LPS+sCD14 or LPS alone was low. LPS+sCD14-induced NF-κB p65 translocation, but the NF-κB response to r-ceramide or C6 ceramide was weak (Figure 4 B, D). For a broader field of view see Figure S6B.
Panels A and C show IRF3 and panels B and D show NF-κB p65 translocation in 70% confluent A549 cells exposed to r-ceramide (SMase (1U/ml), C6 ceramide (30 µg/ml) or LPS+sCD14 (10+1 µg/ml) for 90 min (mean ± SEM of 50 cells/sample, *** p<0.001 between different stimulations, ¤¤ p<0.01, ¤¤¤ p<0.001 compared to control). Panel E shows that p38-MAPK inhibition reduces the IL-8 response to r-ceramide and panels F and G show that p38-MAPK inhibition reduces the nuclear translocation of IRF3 but not NF-κB in A549 cells. Panel H shows that CREB phosphorylation is reduced by p38 inhibition in A549 and A498 cells pre-treated with SB202190 (30 min, 20 µM) and stimulated with r-ceramide (SMase (1 U/ml).
Signaling through p38 MAPK has previously been shown to stimulate proinflammatory responses, including IL-8, IL-6 and TNF [47], [48], [49]. The activation of MAP3K1 and MAP2K3 by r-ceramide exposure of A549 cells suggested that this pathway might be involved upstream of IRF3. Pretreatment of the cells with a p38 inhibitor (SB202190) reduced the IL-8 response to r-ceramide (Figure 4E) and prevented nuclear translocation of IRF3 (Figure 4F, G). NF-κB p65 translocation was not affected by the p38 inhibition (Figure 4F, G). The results suggest that ceramide/TLR4 activates IRF3- rather than NF-κB-dependent transcription, and that the IRF3 response involves p38 MAPK-dependent mechanisms. CREB-phosphorylation was also markedly reduced after p38 inhibition, as shown by confocal microscopy (>99% in A498 cells, >50% in A549 cells, Figure 4H) and Western blots (Figure S7).
Previous work has suggested that the phosphorylation of TRAM is mediated by PKC-epsilon, which is activated downstream of TLR4 [50]. Given that PKC-epsilon is also essential for IRF3 activation, this pathway was examined, using the pan-PKC inhibitor Bisindolylmaleimide II. The inhibitor reduced the response to PMA, which was used as a PKC dependent, positive control. In contrast, the response to ceramide was not impaired (Figure S8).
To further examine the relationship of the ceramide/TLR4 pathway to the classical IRF3 activation pathway, cells were transfected with TBK1 siRNA and responses were compared to irrelevant siRNA transfected cells. In parallel, the cells were transfected with TLR4 and TRAM siRNAs (Knock down efficiency for TLR4 and TRAM was >90% and 64% for TBK1, Figure S9). IRF3-P responses to r-ceramide were reduced by the TLR4 and TRAM siRNAs but were less affected by suppression of TBK1 expression. The response to LPS+sCD14 showed a similar pattern (Figure S9).
These results suggest that the pathway of IRF3 activation identified here has several new features, including p38 dependence and PKC independence. The involvement of TBK1 needs further study.
Bacterial fimbriae and IRF3 translocation in infected human renal tubular epithelial cells
To examine if the IRF3 response is triggered in a pathogen-specific manner involving P fimbriae, we stimulated primary cultures of human CD14+ renal tubular epithelial cells (HRTEC) with isogenic P-fimbriated (E. coli S1918pap) or type 1-fimbriated (E. coli S1918fim) E. coli strains and examined IRF3 by confocal microscopy. Non-fimbriated E. coli S1918 was used as a control. E. coli S1918pap induced higher nuclear IRF3 translocation and IRF3 phosphorylation than E. coli S1918, consistent with results in ceramide-stimulated cells (Figure 5A and Figure S10, p<0.01 for a broader view). There was less IRF3 translocation in response to E. coli S1918fim or to the non-fimbriated control E. coli S1918. All three strains stimulated an NF-κB response, but NF-κB translocation was higher in cells infected with P-fimbriated E. coli compared to type 1-fimbriated E. coli (Figure 5A, p<0.05). Uninfected cells showed no evidence of nuclear IRF3- or NF-κB translocation. The same phenomenon was observed in A498 kidney epithelial cells (Figure S11). In addition, preliminary Western blot analysis of IRF3P in infected cells suggested that S1918pap and S1918fim stimulated a higher response than S1918 (Figure S11).
Panel A shows nuclear translocation of IRF3 and NF-κB in human kidney cells infected with P-fimbriated E. coli (109 CFU/ml, for 90 min). E. coli S1918 was transformed with recombinant plasmids pPIL 110-75 encoding P-fimbriae, (S1918pap) or PKL-4 encoding type 1 fimbriae (S1918fim). Infection increased nuclear staining above background for all three strains. E. coli S1918pap triggered stronger IRF3 and NF-κB responses than E. coli S1918 or E. coli S1918fim. Quantifications of nuclear fluorescence intensity are given in the table, means ± SEMs of 30 cells/sample; for a broader view see Figure S11. Panel B shows IRF3 target gene expression after stimulation of A498 kidney epithelial cells with E. coli CFT073 (Illumina whole genome microarrays, figure shows fold change above uninfected control cells, ≥log 2 cut off). Infection stimulated genes in the IFN pathway and increased the IFNβ response. Panel C shows TLR-specific superarray data. Panel D shows the increased susceptibility of Ifnβ−/− mice to infection with CFT073, compared to wt mice. Bacterial numbers are compared in urine and tissues (Kidneys, bladders and spleens).
Increased susceptibility to UTI in Ifnβ−/− mice
IRF3 target gene expression was examined by microarray analysis. We infected A498 kidney epithelial cells in vitro with virulent CFT073 or non-pathogenic E. coli (4 hours, 108 CFU/ml) and complementary RNA was hybridized to Illumina whole genome microarrays. There was a dramatic IFNβresponse to infection (24-fold above uninfected control cells, ≥log 2 cut off) (Figure 5B). By Ingenuity Pathway analysis, we detected significant activation of several members of the interferon-signaling pathway such as IFIT1, STAT1 ISG15, IP-10 and IFNAR2. A weaker ISG15 response was observed (1.8-fold above background). By RT-PCR, a strong IFNβ response to r-ceramide was confirmed in human kidney cells (A498, Figure 5 B and C).
To examine if the effects of IRF3 on host susceptibility are IFNβ-dependent, we infected Ifnβ−/− mice with E. coli CFT073 and examined parameters of disease and bacterial persistence (Figure 5E). Bacterial clearance was drastically impaired in Ifnβ−/−mice compared to wt controls (Figure 5E, p<0.001 between Ifnβ−/−and wt mice in urine, kidneys and bladders). The Ifnβ−/− mice also developed abscesses. Positive spleen cultures confirmed systemic spread of infection in these mice, which also developed symptomatic disease and were sacrificed on day three (Figure 5E). The results suggest that IFNβ is activated by infection and that IFNβ might be an essential effector molecule in IRF3-dependent bacterial clearance.
IRF3 promoter polymorphisms in patients with urinary tract infection
To examine if UTI susceptibility is associated with differences in IRF3 promoter efficiency, IRF3 promoter sequence variation was studied in two highly UTI-prone patient populations. Sample 1 comprised children in southern Sweden, with a consistent UTI pattern over several years: either severe recurrent kidney infections (APN; n = 21) or asymptomatic carriage of E. coli with no prior symptomatic infection (primary asymptomatic bacteriuria, ABU, n = 16). These children were identified after prospective, long-term follow-up of a larger patient group. Sample 2 comprised adults in western Sweden, with a history of childhood UTI (n = 82). They were enrolled in a prospective study of febrile UTI (APN) in the 1970s and were recently re-evaluated, after about 30 years, to investigate UTI morbidity and long-term effects on health and kidney function. Both samples included additional patients who developed ABU secondary to an APN episode (secondary ABU, n = 16 in sample 1 and n = 61 in sample 2). Controls were children without UTI or related morbidity (n = 27) and adult blood donors (n = 62) from the same areas.
DNA sequencing of IRF3 promoters from UTI patients revealed variation at the −925 and −776 positions. SNPs −925 and −776 were linked in the study population (r2 = 1.0) but the IRF3 genotype varied with UTI severity (Figure 6A–B). Genotype counts for −925 and −776 were in Hardy Weinberg Equilibrium across both case and control samples apart from the APN group (χ2 = 47, p<0.001), indicating effects of genetic drift in the APN group. We observed significant differences for the two studied markers between cases and controls in allelic or genotypic models. In sample 1, most of the APN patients were homozygous for the two positions (A/A–C/C, 79% vs. 25% in primary ABU, Figure 6A p = 0.0017). The results in APN patients were confirmed in sample 2, with 75% homozygous and 13% heterozygous SNPs compared to 53% and 37% in adult controls. The differences were confirmed when the two samples were combined, as shown in Figure 6B. Furthermore, the minor allele frequency was decreased in APN compared to primary ABU (p = 0.0103) and controls (p = 0.0239) (Figure 6B). The minor allele frequency for paediatric UTI patients, adult UTI patients and the relevant controls are demonstrated in Supplemental Table S4 in Supporting Information S1.
Panel A shows the IRF3 promoter polymorphisms (−925 and −776) in highly selected, UTI-prone pediatric and adult patients compared to healthy controls. The homozygous, A/A, C/C genotype at positions −925 and −776 dominated in pyelonephritis-prone patients, while the G/A, C/T heterozygous genotype was more common in the asymptomatic carriers. Panel B presents genotype and allele counts for markers −925 and −776 with samples 1 and 2 combined. Panel C shows the IRF3 promoter activity in transfected human kidney epithelial cells. IRF3 promoter SNPs representing the ABU and APN genotypes were amplified from patient DNA altered by site-directed mutagenesis and cloned into a luciferase reporter plasmid. The predominating APN genotype (A-C) lowered the luciferase expression levels compared to the ABU genotype (A-T). Graphs show means of one representative experiment out of three, each done in triplicate.
The IRF3 genotype of the secondary ABU patients resembled the APN groups in both samples, consistent with their prior APN episodes.
The acute pyelonephritis-associated promoter SNPs reduced transcription efficiency
To examine if the IRF3 promoter variation influences transcription efficiency, we cloned promoters from one patient with APN and one with ABU into a luciferase reporter vector. We then changed the APN haplotype at positions −925, −776 to the predominating ABU haplotype (A-C to G-T), or the ABU haplotype to the APN haplotype (G-T to A-C) by site-directed mutagenesis. We then transfected A498 human kidney epithelial cells with the different promoter constructs and determined luciferase activity (Figure 6C). The promoter was functional in these cells, resulting in luciferase activity above the vector control.
Transcriptional activity from the APN promoter (A-C) was about 50% lower compared to the ABU (A-T) promoter (Figure 6C). This difference could be attributed to the polymorphic sites, as the promoter activity increased when the APN A-C haplotype was mutated to G-T and decreased when the ABU G-T haplotype was mutated to A-C (p<0.001). This difference was confirmed by cloning the IRF3 promoters from three additional APN (A-C) and three ABU (G-T) patients (p = 0.001). The results show that the IRF3 promoter efficiency is reduced by the SNPs occurring in about 80% of APN patients, consistent with the human SNPs reducing IRF3 expression and increasing the risk for APN.
IRF3 was originally described as a transcription factor controlling interferon responses to viral infection [51]. More recently, the involvement of IRFs in antibacterial defense and immunoregulation by TLRs has received more attention, since NF-κB, IRF3 and AP-1 form transcriptional complexes that regulate innate immune responses in monocytes [52]. The relevance of IRF3 to human pathology has not been investigated, however. We show that IRF3 is activated in a pathogen-specific manner by P-fimbriated, uropathogenic E. coli, through a new signaling pathway involving TLR4, TRAM, CREB and p38. In the absence of IRF3, acute morbidity and extensive tissue damage are dramatically augmented, consistent with the need for this pathway to maintain a functional antimicrobial defense. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response.
Mucosal pathogens exploit the extracellular domains of sphingolipids as receptors for AB toxins such as Shiga and cholera toxin, as well as attachment ligands for Pseudomonas aeruginosa, HIV gp120 and uropathogenic E. coli [53], [54], [55]. This study provides evidence that P-fimbriated E. coli, SMase and exogenous, free ceramide all activate the IRF3-dependent innate immune response. Soluble, exogenous ceramide and SMase were used in parallel in these experiments to ensure that the synthetic, short-chained form of ceramide and the endogenous, long-chained form adequately represented the membrane-anchored species in intact cells. SMase is contaminated by low amounts of LPS, but these trace amounts were insufficient to activate an innate immune response in the CD14-negative mucosal cells used in this study [9], [14]. FRET analysis showed that ceramide is approximated to TLR4 in the cell membrane, suggesting that a direct interaction with TLR4 and/or the early adaptors trigger this pathway. Such ceramide-induced TLR4/IRF3 signaling might offer a general mechanism for host sensing of pathogens that perturb membrane sphingolipids in mucosal cells.
To provide further evidence that ceramide signaling via TLR4 to IRF3 discriminates virulent from commensal bacteria, we infected Irf3−/− mice with a commensal-like E. coli strain from a patient with asymptomatic bacteriuria. This strain did not trigger a significant response and was cleared efficiently, suggesting that the IRF3 pathway was not alerted. In constrast, a P-fimbriated transformant triggered rapid, septic infection in the Irf3−/− but not in wt mice linking this virulence factor that recognizes glycosphingolipid surface receptors, to the IRF3-dependent host response. In contrast, IRF3 was not activated by a type 1 fimbriated isogenic strain, suggesting a preference for glycosphingolipid rather than glycoprotein receptors. This does not negate the previous finding that FimH acts as an immune inducer, protecting against viral infection associated with TLR4 and type 1 interferon signaling, but suggests that the mechanisms differ [56]. The results confirm the pathogen specificity of the IRF3 response and the role of P fimbriae as a virulence ligand triggering this response. As a consequence of this selective IRF3 activation, the uropathogenic or P-fimbriated, commensal E. coli strains influenced epithelial gene transcription in a pathogen-specific manner.
IRF3 phosphorylation in response to ceramide was controlled by TLR4 and TRAM, as shown using specific siRNA knock down. Activation was not TBK1 or PKC dependent, however, suggesting alternative activation compared to previously described mechanisms of IRF3 activation [50]. A schematic of the identified kinases and targets is given in supplemental Figure S4. Although this signaling pathway has not been entirely deciphered, a strong involvement of TRAM and CREB was detected as well as involvement of p38 MAPK-dependent events. In this model, IRF3 activation was not controlled by PKC dependent mechanisms, however. The involvement of TBK1 is not clear, but preliminary experiments did not provide evidence that TBK1 controlled IRF3 phosphorylation in this pathway. In addition, P-fimbriated E. coli strains and ceramide significantly activated NF-κB, thus providing a broad basis for the innate immune response to the intact, complex pathogen. Importantly, the IRF3 response differed after LPS+sCD14 stimulation, further suggesting that pathogen recognition and pattern recognition agonists trigger partially different signaling pathways.
The phenotype of Irf3−/− mice predicted that reduced IRF3 expression could also increase human susceptibility to severe kidney infection. In support of this hypothesis, there were marked promoter sequence differences between children with ABU or APN in a long-term prospective study and we confirmed an association of polymorphisms to disease severity in adult patients who were followed for about 30 years after their first febrile UTI episode. In the past, we have shown that genetic variation affecting innate immunity modifies human UTI susceptibility [2]. Chemokine receptor expression and neutrophil function are modified by CXCR1 expression, and promoter variants reducing TLR4 expression are coupled to asymptomatic bacteriuria [22], [57], [58], [59], [60]. The present study adds IRF3 to this short list of polymorphic innate immune response genes that distinguish asymptomatic carriers from APN-prone patients.
The human IRF3 promoter has a number of transcription factor binding sites, including a HOX box, three SP1 sites, NF1, USF, SRF and IRF1-like site and functional elements are within a 113-nucletide long fragment, containing one Sp-1 site, the IRF1-like site, NF1 and HOX box. SNP −925 is located within this region, indicating a possible role in promoter efficiency. IRF3 promoter SNPs were first described in patients with systemic lupus erythematosus (SLE) [61]. It was speculated that the A-C haplotype increased IRF3 transcription and that the G-T haplotype might protect against SLE by reducing type I IFN production. The effect of the Irf3 deletion on disease susceptibility in mice suggested, however, that risk might be associated with reduced, rather than increased, IRF3 function. This idea was supported by luciferase reporter assays designed to test the IRF3 promoter sequences typical of APN- or ABU-prone individuals.
UTIs are among the most common bacterial infections in man, and remain a major cause of morbidity and mortality [62], [63]. A subset of disease-prone individuals is at risk for recurrent severe pyelonephritis and renal dysfunction. Therefore, there is a need to identify and treat these patients, preferably in infancy, when many of them experience their first febrile UTI episode. Although predictive diagnostic tools have been suggested [58], [59], [60], the present study identifies IRF3 for the first time as an innate immune response gene involved in UTI. Thus, IRF3 may be a new molecular target in the diagnosis of UTI susceptibility, potentially creating more precise approaches for detection and prevention of severe, recurrent kidney infection and associated debilitating morbidity.
Ethics statement
For research involving humans, informed written consent was obtained from all participants or their parents/guardians. The study was approved by the Ethics Committee of the medical faculty, Lund University, Sweden (LU106-02, LU236-99).
All the animal experiments were performed with the permission of the Animal Experimental Ethics Committee at the Lund District Court, Sweden (numbers M166-04 and M87-07). Experimental UTI was performed in a level P2 biohazard laboratory within the MIG animal facility and was governed by the following directive, law, ordinance and provisions: Council Directive EG 86/609/EEC, the Swedish Animal Welfare Act (Djurskyddslag: 1988:534) and the Swedish Animal Welfare Ordinance (Djurskyddsförordning: 1988 :539). Provisions regarding the use of animals for scientific purposes: DFS 2004:15, DFS 2005::4, SJVFS 2001:91, SJVFS 1991:11.
SMase (Staphylococcus aureus), bovine serum albumin, SDS, LPS (Salmonella typhimurium), C6 ceramide, SB202190 and Bisindolylmaleimide II were from Sigma Aldrich, St Louis, MO, USA. Soluble CD14 (sCD14) was from Biometec, Greifswald and IL-8 was quantified by Immulite 100, Siemens, Germany. Lipofectamine 2000 transfection reagent was from Invitrogen. siRNA downregulation (Supplemental Table S1 in Supporting Information S1) was validated by qRT-PCR, using primers: TLR4 (Hs00152939, Applied Biosystems), MyD88 (QT00203490, Qiagen), TRAM (QT00033341, Qiagen), TBK1 (QT00078393). TBK1 siRNA (sc-39058), a pool of 3 target-specific 19–25 nt siRNAs was from Santa Cruz Biotechnology (USA). Human Phospho-Kinase Array Kit ARY003 was from R&D Systems, Abingdon, Oxford, UK. Transcriptome analysis of r-ceramide or LPS+sCD14 activated cells (1 or 3 hours) was by Superarray (PAHS018, SaBioscience) for 84 TLR signaling pathway genes. IRF3 promoter SNPs were identified by Pyrosequencing using the PSQ 96 SNP Reagent Kit (Biotage, Uppsala, Sweden, Supplemental Table S1 in Supporting Information S1). Rabbit anti-human TLR4 primary antibodies were from eBioscience, CA, USA, mouse anti-ceramide primary antibodies, clone MID 15B4 from ALEXIS Corporation, Lausen, Switzerland. Rabbit anti-human primary antibodies against CREB-P (Ser 133), Fos-P (Thr 232), JNK-P (Thr 183/Tyr 185) and IRF3 and mouse anti-human-NF-κB p65 antibodies from Santa Cruz Biotechnology (USA), rabbit anti-IRF3-P (Ser 396) antibodies were from Cell Signaling Technology. Rabbit anti-human-TRAM-P (raised against the N-terminal end of the protein, aa 7–21 containing phosphoSer at aa 16) and TRAM antibodies were from FabGennix Inc., Frisco, TX, USA, NIMP-R14 rat anti-mouse neutrophil specific antibodies from Abcam, Cambridge, USA, polyclonal rabbit antiserum raised against a peptide within the PapG adhesin (CRPSAQSLEIKHGDL) was used to detect P-fimbriated E. coli. Alexa 488 anti-rat IgG, Alexa 568 anti-rabbit IgG, Alexa 568 anti-mouse IgM and Alexa 488 anti-mouse-IgG secondary antibodies were from Invitrogen, Eugene, Oregon, USA. Swine anti-rabbit immunoglobulins-HRP secondary antibodies were from DAKO A/S, Glostrup, Denmark and Santa Cruz Biotechnology (USA). FRET and fluorescence microscopy was by LSM510 META confocal microscope (Carl Zeiss, Oberkochen, Germany).
Cell cultures
The human lung adenocarcinoma A549 (ATCC CCL-185) and kidney carcinoma A498 (ATCC HTB-44) epithelial cell lines were grown in RPMI 1640 supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids, 50 µM/ml gentamicin, and 5% FBS. Human renal tubular epithelial cells (HRTEC) were isolated as described [64]. Cells were maintained at 37°C+5% CO2 in a humidified atmosphere, split weekly and exposed to P-fimbriated, type I fimbriated or non-fimbriated E. coli, 0.1–1 U/ml of SMase, freshly prepared C6 ceramide or LPS+sCD14. IL-8 secretion was quantified by Immulite 100 (Siemens, Bad Nauheim, Germany).
LSM 510 Meta confocal laser-scanning microscopy was used for FRET acceptor photobleaching and imaging of epithelial cells. Cell stimulation/infection was in 8-well chamber slides (LabTek, Nunc, RPMI+5% foetal calf serum). The cells were first stimulated with r-ceramide (1U of SMase/ml) or LPS+sCD14 (10+1µg/ml), fixed with 3.7% formaldehyde and stained with a mouse antibody specific for native ceramide (sphingosine-trans-D-erythro-2-amino-4-octadecene-1.3-diol) and with a rabbit polyclonal antibody, specific for the extracellular domain of TLR4 (amino acids 6–169). Secondary antibodies to TLR4 and free ceramide were conjugated with Alexa-488 (donor) and Alexa-568 (acceptor), respectively. FRET efficiency was estimated in percent of fluorescence increase calculated by: FRET efficiency = ((IDA-IDB)/IDA)×100% where IDA is the donor intensity after bleaching and IDB the donor intensity before bleaching.
siRNA transfection
A549 human epithelial cells in 24-well plates (TPP) were siRNA transfected, using Lipofectamine 2000 (Supplemental Table S1 in Supporting Information S1). Knockdown efficiency was validated by qRT-PCR. After a 72 h incubation, transfected cells were stimulated with SMase (1 U/well) or LPS+sCD14 (10+1µg/ml). Supernatants were collected after 24 h.
RT-PCR array
Total extracted mRNAs were converted to cDNA using RT2 First Strand Kit (SABioscience Corporation, Fredrick, MA, USA). The transcriptomic profile of cells exposed to r-ceramide or LPS+sCD14 was examined using a RT-PCR-based superarray, containing 84 genes involved in TLR signaling (SABiosciences, PAHS018). Gene expression levels were calculated by the ΔCt method and normalized to five housekeeping genes. RT-PCR was used to determine the efficiency of siRNA knockdown. The TaqMan system was used to quantify TLR4 and GAPDH cDNA and the QuantiTect SYBR Green systems was used to quantify other genes of interest. cDNA was quantified by RT-PCR using a Rotor gene 2000 instrument (Corbett Life Science, Sydney, Australia) and normalized against GAPDH.
Phospho-kinase array
Protein phosphorylation was quantified using the Human Phospho-Kinase Array Kit (Proteome Prolifer Array, R&D Systems, Abingdon, Oxford, UK). Protein extracts were prepared from 100% confluent A549 cells cultured in 6-well plates and treated with 1U/well SMase or LPS+sCD14 (10+1µg/ml). Untreated cells were used as control. The signals were detected with the ECL Plus Western Blotting Detection System (GE Healthcare).
SDS-PAGE and immunoblotting
In order to detect phosphorylated TRAM, A549 cells grown in 6-well plates were stimulated with 1U/mL SMase, 0.1 µg/mL LPS+sCD14 (10+1µg/ml), 15 µg/mL C6 (Sigma) or RPMI medium alone for 45 and 90 min. Cells were lysed in ice-cold buffer (10 mM HEPES-KOH, 5 mM EDTA, 0.5% Nonidet P-40 and 10 mM KCl, pH 7.9) containing a protease inhibitor mix (Complete, Roche Diagnostics GmbH, Mannheim, Germany) and 1mM Na3VO4. After 10 min incubation, lysates were centrifuged for 5 min at 12000 g at 4°C and protein concentrations in the collected supernatants were quantified using the DC protein assay kit (Bio-Rad, Hercules, USA). Proteins in the lysates were separated by SDS-PAGE (4–12% NuPAGE Bis-Tris gels, Invitrogen) on ice with NuPAGE MES SDS running buffer (Invitrogen). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using NuPAGE transfer buffer (Invitrogen) and the membrane was probed with phospospecific primary antibodies followed by HRP-labeled, swine anti-rabbit IgG. Bound antibodies were visualized with the ECL Plus Western Blotting Detection System.
Native PAGE and immunoblotting
IRF3 dimerization was detected by native PAGE and immunoblotting. A549 cells cultured in 6-well plates were stimulated with medium alone, 1 U/mL SMase and 0.1 µg/mL or LPS+sCD14 (10+1µg/ml), for 90 min. Whole cell lysates in a buffer containing 50 mM Tris HCl, pH 7.5, 400 mM NaCl, 1mM EDTA, 1% Nonidet P-40 were separated by electrophoresis on a 7.5% native Tris-glycine gel [65]. Membranes were incubated with primary antibodies against human IRF3 (Fl-425, Santa-Cruz) diluted 1∶1000 and anti-rabbit IgG-HRP (1∶1000). IRF3 monomers and dimers were detected with the ECL Plus Western Blotting Detection System.
DNA microarray analysis
In brief, A498 cells (n = 350000) were seeded in 6-well plates and infected with CFT073 (108 CFU/ml), total RNA was extracted (Trizol, Invitrogen, USA) and cleaned by a Qiagen RNeasy. RNA was reverse-transcribed to biotin-labeled cRNA using a TargetAmp Nano-g Biotin-aRNA Labeling kit (Epicentre Biotechnologies, Madison, USA). Labeled cRNAs were hybridized onto an Illumina HumanHT-12 Expression Beadchip for 16 hours at 58°C. The arrays were then washed and stained (Illumina Wash Protocol) and scanned using a BeadArray Scanner 500GX. The background-subtracted data were pre-processed to correct negative and non-significant intensities. Pre-processed data was normalized using the cross-correlation [66] and genes with a log fold change of 2 were identified as differentially expressed. Data was preprocessed using RMA implemented in the free software packages R and Bioconductor (http://www.r-project.org). For more details, see Yadav et al.
Differentially expressed genes were categorized using the Functional Annotation Clustering Tool in the Database for Annotation, Visualization and Integrated Discovery (DAVID) [67] and the EASE score (a modified enrichment score derived from Fisher exact P-value) was used to judge the enrichment. To further study signaling pathways altered by CFT073, the differentially expressed genes were submitted for Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA).
Bacteria and growth conditions
Escherichia coli S1918 [68] (KanR) lacks genes encoding known adhesins and was used as a recipient strain for recombinant plasmid pPIL 110-75 (AmpR) carrying the papAD1100 gene cluster (pap+) [69] or PKL-4 carrying the entire fim gene cluster from E. coli PC31 (type I+) [68]. The papGX deletion mutant of E. coli strain 83972 (ABU 83972ΔpapGX) was generated using the lambda red homologous recombination technique [70]. Clones with the reconstituted pap determinant were screened using PCR and verified by DNA sequencing. Primers used for reconstitution of a functional pap gene cluster in E. coli 83972 are shown in Supplemental Table S5 in Supporting Information S1. The growth rates of the reconstituted mutant strain and the 83972 wild type strain were shown to be identical. In addition, the ability of the reconstituted mutant to agglutinate sheep blood erythrocytes was also confirmed by agglutination assays.
Experimental urinary tract infection (UTI)
Mice were bred at the MIG animal facilities, Lund, Sweden. Female C57BL/6 wild type or Irf3−/− (from T. Taniguichi) and Ifnβ−/− (from F. Ivars, Lund University) mice were used at 9–15 weeks. After anesthesia (Isofluorane), mice were infected by intravesical inoculation with E. coli CFT073 (109 CFU in 0.1 mL) through a soft polyethylene catheter (outer diameter 0.61 mm; Clay Adams, Parsippany, NJ, USA). Animals were sacrificed while under anesthesia, kidneys and bladders were removed and prepared for hematoxylin-eosin staining or immunohistochemistry. Viable counts in homogenized tissues (Stomacher 80, Seward Medical, UAC House, London, UK) were determined after overnight growth on tryptic soy agar plates at 37°C. Urine samples collected prior to and daily after infection were cultured and recruited neutrophils were quantified in uncentrifuged urine by use of a hemocytometer.
Histology and immunostaining
Kidney sections were examined by immunohistochemistry [71]. Tissue sections were dried and permeabilized in 0.2% Triton X-100, 5% goat normal serum (DAKO) in PBS, incubated with NIMP-R14 rat anti-mouse neutrophil specific antibodies (1∶200) and a polyclonal rabbit antiserum to the Pap G adhesin (1∶200) to detect P-fimbriated E. coli and to Alexa 488 anti-rat IgG and Alexa 568 anti-rabbit IgG secondary antibodies and nuclei were counterstained with DAPI (0.05 µM). After mounting, coverslipped slides were examined by fluorescence microscopy (AX60, Olympus Optical, Hamburg, Germany) at the Department of Pathology, Lund University, Sweden.
Confocal fluorescence immunocytochemistry was performed on cells grown to 70–80% confluence on 8-well chamber slides. After stimulation, cells were fixed and permeabilized with 0.25% Triton X-100, 5% FBS in PBS and incubated with primary antibodies diluted 1∶50 in 5% FBS in PBS overnight at 4°C. Alexa fluor-labeled secondary antibodies were applied for 1 hour at RT in the dark. In order to control specific staining of neutrophils and bacteria, slides were stained with only secondary antibodies (Figure S1). Slides were covered with mounting medium (M1289, Sigma) and cover glasses and the cells were examined with a LSM510 META confocal microscope.
UTI-prone patients and pyrosequencing
The IRF3 promoter from patients with UTI or healthy controls was sequenced (PSQ96, Biotage, Uppsala, Sweden) and examined for −925 and −776 polymorphisms [61]. Genomic DNA was extracted from heparinized peripheral blood using the QIAamp DNA Blood midi kit. More detailed descriptions of inclusion critera and diagnosis are provided in [58], [59], [72]. The IRF3 promoter SNPs (−925; −776) were genotyped using Pyrosequencer PSQ 96 after PCR amplification of chromosomal DNA and a second biotinylated PCR for each SNP (for primers see Supplemental Table S2 in Supporting Information S1).
Transient transfection and dual luciferase reporter assay
The promoter sequences from extracted chromosomal DNA derived from APN and ABU patients were PCR-amplified using the Infusion primers 5′ IRF3 NheI and 3′IRF3 NcoI (Supplemental Table S3 in Supporting Information S1) and Phusion hot start polymerase according to the manufacturer (Finnzymes Oy, Finland). Amplicons were introduced by recombination, using the Infusion cloning technique (Clontech), into a NheI- and NcoI-cleaved and gel-purified luciferase reporter vector, pGL3 basic (Promega). The recombinant DNA was transformed into E. coli and recombinant clones were screened for the presence of cloned promoter insert. Plasmids of the correct size were further analyzed by DNA sequencing using the Big Dye terminator v3.1 cycle sequencing chemistry and ABI capillary sequence.
A quick change Multi Site-directed Mutagenesis kit (Stratagene) was used according to the manufacturer's instructions to create the various IRF3 promoter constructs (Supplemental Table S3 in Supporting Information S1). 5498 human kidney epithelial cells were cultured in 24-well plates at a density of 2.5×105 cells per well. The cells were transiently transfected with wild type or mutant IRF3 promoter driven firefly luciferase constructs (pGL3) together with a constitutively expressed internal control construct with Renilla luciferase-thymidine kinase promoter (pRL-TK, Promega) using Fugene HD (Roche) Transfection reagent at 4∶2 ratio. Luciferases were measured using the Dual Luciferase Reporter System Assay (Promega) with a Glomax Integrated Luminometer (Promega). Firefly luciferase data were normalized against transfection efficiency of Renilla luciferase and expressed as a ratio.
Student's t test or Wilcoxon's rank-sum test were used for paired comparisons, Mann-Whitney test was applied for unpaired comparisons. P values below 0.05 were considered to indicate statistical significance. Deviations from Hardy-Weinberg equilibrium (HWE) for genotypes at individual loci in patients and controls, as well as differences in genotype and allele distributions between groups, were assessed using the χ2 test. Fisher's exact test was used where appropriate.
List of ID numbers for genes and proteins of mouse and humans
Gene ID number for human TLR4 is 7099, human MyD88 is 4615, human TRIF is 148022, human TRAM is 353376, human TBK1 is 29110, human CREB is 1385, human IRF3 is 3661, mouse Irf3 is 54131, human IFNB is 3456 and mouse Ifnb is 15977.
Supporting Information
Figure S1.
Secondary antibody control of kidney sections containing neutrophils and P fimbriated E. coli. Hematoxylin/Eosin staining of abscesses and a corresponding area stained with only secondary goat anti-rat immunoglobulins, conjugated with Alexa fluor-488 and secondary goat anti-rabbit immunoglobulins, conjugated with Alexa fluor-568.
Figure S2.
qRT-PCR analysis of knockdown efficiency after siRNA transfection. The knockdown of TLR4, TRAM and MyD88 expression in A549 cells was confirmed by RT-PCR. The mRNA levels were coamplified using GAPDH mRNA as an internal standard. Cells transfected with an irrelevant siRNA (Ctrl) were used as a control. Suppression of TLR4 mRNA and TRAM mRNA was more than 90%, MyD88 mRNA was downregulated by 80%.
Figure S3.
Broader field of view of TRAM phosphorylation (TRAM-P) after 30 or 60 minutes of r-ceramide (SMase (1U/ml), C6 ceramide (30µg/ml) or LPS+sCD14 (0.1+1 µg/ml) exposure (primary polyclonal rabbit-TRAM-P antibodies and secondary anti-rabbit-Alexa fluor-568 labelled antibodies).
Figure S4.
Identified responders in ceramide/TLR4 induced signalling; a simplified model. P fimbriated Escherichia coli use glycosphingolipid receptors to adhere to uroepithelial cells. Binding triggers ceramide release followed by TLR4 and TRAM activation. Downstream signaling involves MAP kinases, CREB, IRF3 nad AP-1 (Jun/Fos). CREB and IRF3 phosphorylation is partly p38 MAPK dependent but not dependent on PKCε and TBK-1.
Figure S5.
Genes involved in TLR4 signaling. Panel A shows A549 epithelial cells, stimulated for 1 h with r-ceramide (SMase, 1U/ml) or LPS+sCD14 (10+1µg/ml). Panel B shows A498 epithelial cells stimulated for 3 hours. Relative gene expression was analyzed by RT-PCR-based superarray.
Figure S6.
Panel A shows IRF3 staining in J82 human bladder epithelial cells. J82 cells were exposed to r-ceramide (SMase (1U/ml)), LPS (0.1 µg/ml) or LPS+sCD14 (0.1+1 µg/ml) for 90 min and analyzed as described in figure 4A. N = Nuclear staining. Panel B shows Nuclear IRF3 translocation in response to ceramide/TLR4 in A549 cells. IRF3 and NF-κB p65 translocation in 70% confluent A549 cells exposed to r-ceramide (SMase (1U/ml), C6 ceramide (30 µg/ml) or LPS+sCD14 (10+1 µg/ml) for 90 min. N = Nuclear staining.
Figure S7.
r-ceramide induced CREB and IRF3 phosphorylation in mouse renal tubular cells (MRTEC) was reduced after treatment with a p38 inhibitor (SB202190). MRTECs were stimulated for 90 min with r-ceramide (SMase, 1U/ml) or LPS+sCD14 (0.1+1µg/ml). Blots of whole cell extracts were stained with phosphospecific rabbit anti-CREB-P- or rabbit anti-IRF3-P- and HRP-conjugated anti-rabbit antibodies. The western blot is a representative of 2 experiments.
Figure S8.
Interleukin-8 (IL-8) secretion in A549 cells after treatment with a PKC inhibitor (Bisindolylmaleimide II, 1300 nM) and 24 hours stimulation with r-ceramide (SMase, 2 U/ml), LPS+sCD14 (0.1+1 µg/ml) or PMA (0.01 ng/ml). Means ± SEM of two independent experiments. Med = Medium alone.
Figure S9.
Knockdown of TLR4 and TRAM results in abrogation of the ceramide dependent activation of IRF3 phosphorylation while knock down of TBK-1 does not. Western blot analysis after siRNA transfection in A549 cells of TLR4, TRAM or TBK1 siRNA, irrelevant siRNA was used as a control. The knockdown of TLR4, TRAM and TBK1 genes were confirmed by RT-PCR. The knockdown efficiency was more than 90% for TLR4 and TRAM, and 64% for TBK1.
Figure S10.
Broader field of view of nuclear translocation of IRF3 and NF-κB in primary human renal tubular epithelial cells after stimulation E. coli (N = Nuclear staining, B = Bacteria). The P-fimbriated strain (E. coli S1918pap) induced higher nuclear IRF3 translocation than non-fimbriated (S1918) and Type 1 fimbriated (S1918fim) E. coli while NF-κB was translocated in response to all strains, although slightly more in P-fimbriated E. coli.
Figure S11.
Panel A represents confirmatory experiment of data described in Figure 5A. P-fimbriated E. coli (S1918pap) induces IRF3 translocation in A498 human kidney epithelial cells more efficiently than cells stimulated with unfimbriated E. coli (S1918) and type 1 fimbriated E. coli (S1918fim) N = Nuclear staining. Panel B - Western blotting showed higher IRF3-P activation in the S1918pap infected A498 cells.
Supporting Information S1.
Tables S1 to S5.
C57BL/6 Irf3−/− mice were generously provided by Professor T. Taniguchi, University of Tokyo, Japan. We thank Josephine Lum and Henry Yang at Singapore Immunology Network (SIgN), A*Star, Singapore and Sandra Andersson and Linda Åkesson at Department of MIG, Lund University for excellent technical assistance.
Author Contributions
Conceived and designed the experiments: HF CS. Performed the experiments: HF NL BR MY KJ AU AAH SR PS. Analyzed the data: HF NL BR MY KJ AU AAH SR CS. Contributed reagents/materials/analysis tools: UD ES DK UJ CS. Wrote the paper: HF NL BR CS.
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Kunin C (1987) Urinary tract infections. Detection, prevention and management, 5th ed. Baltimore: Williams & Wilkins.
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- 72. Martinell J, Jodal U, Lidin-Janson G (1990) Pregnancies in women with and without renal scarring after urinary tract infections in childhood. Br Med J 300: 840–844.


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National FJ Summit Recap
Every July the Toyota 4×4 community has been meeting up for a 5 days of trail riding and camaraderie in the beautiful landscape of Ouray, Colorado for the National FJ Summit. This year marked the 10th annual event and FJ Summit X didn't disappoint. Open to more than just FJ Cruisers, this event has Toyotas of all […]
BDS New Product Announcement #251: '01-10 GM HD RECOIL Traction Bars
The latest BDS RECOIL Traction Bars to hit the market are designed specifically for the 2001-2010 Chevy and GMC 2500/3500 HD trucks with a range of lift heights and easy installation.
2016 PA Jeeps All Breeds Show Recap
Each summer the team from BDS Suspension spends a fair amount of time in the great state of Pennsylvania. Between the truck shows, Jeep shows, races and dealer stops there is no shortage of activities. Two weekends ago we attended the 4Wheel Jamboree Nationals in Bloomsburg, PA followed by another trip back this past weekend for […]
BDS heads to Bloomsburg for the 4Wheel Jamboree Nationals
This past weekend BDS made the trip east to Bloomsburg, PA for the 4Wheel Jamboree Nationals. This annual event attracts thousands of 4×4 enthusiasts from around the Midwest for 3 days of fun in the sun. This year's activities included monster truck racing and freestyle, show-n-shine competition, tough truck racing, miss-4wheel jamboree bikini contest, FOX […]
BDS New Product Announcement #250: NX2 Nitro Series Shocks Included in BDS Lift Kits
BDS Suspension will now include 8500-series NX2 Nitro Premium Shock Absorbers as standard equipment in all (non-FOX coilover system) BDS lift kits. Designed for improved performance and control, these new shocks use vehicle specific valving and offer better dampening capabilities on lifted vehicles. They use a nitrogen charged twin-tube design with velocity sensitive valving for lightning fast adaptive dampening to whatever terrain you traverse.
BDS New Product Announcement #249: Air Ride RAM 2500 Lift Kits
BDS Suspension announces the release of 6″ lift options for the RAM 2500 4WD with rear air ride. Available in standard radius arm drop bracket and 4-link conversion setups, these new suspension systems offer improved performance, capability and room for 37″ tires under your 2014-2016 RAM 2500 4wd diesel trucks equipped with factory rear air ride.

subset: commoncrawl


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About the Authors
Stockholm Bioinformatics Center, Center for Biomembrane Research, Stockholm University, Stockholm, Sweden
Corresponding Author
Competing Interests
The authors have declared that no competing interests exist.
Author Contributions
ÅKB and AE conceived and designed the experiments. ÅKB performed the experiments. ÅKB and DE analyzed the data. ÅKB, DE, and AE contributed reagents/materials/analysis tools. ÅKB and AE wrote the paper.


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Lala (Age: 34) + Carmelo Anthony (Age: 29)
One of our favorite celebrity couples, Lala and Carmelo Anthony, have a five year age difference! Lala revealed that when she and Carmelo first met, he lied about his age and claimed was 21 when he was really only 19 years old! Despite Carmelo's age, the two found love and were married in 2010.
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Residence Inn West Orange
107 Prospect Avenue, West Orange, NJ 07052, United States of America – Great location - show map
Excellent location – rated 9.2/10! (score from 29 reviews)
Rated by guests after their stay at Residence Inn West Orange.
|Room Type
|One-Bedroom Suite
|King Studio
|King Studio with Sofa Bed
Make yourself at home at the Residence Inn West Orange. Both business travelers and families will love our convenient hotel location near Montclair, NJ.
Residence Inn West Orange takes special requests – add in the next step!
5 Reasons to Choose Residence Inn West Orange
Low rates
3 properties in West Orange
Manage your bookings online
Booking is safe
Facilities of Residence Inn West Orange Great facilities! Review score, 8.8
Pets are not allowed.
- Fitness Center
Free! WiFi is available in all areas and is free of charge.
Free! Wired internet is available in the hotel rooms and is free of charge.
Free! Free public parking is available on site (reservation is needed).
- Vending Machine (drinks)
- Vending Machine (snacks)
- 24-Hour Front Desk
- Express Check-in/Check-out
- ATM on site
- Meeting/Banquet Facilities
- Business Center
- Fax/Photocopying
- Newspapers
- Safe
- Non-smoking Rooms
- Facilities for Disabled Guests
- Elevator
- Heating
- Air Conditioning
From 3:00 PM
Until 12:00 PM
Cancellation and prepayment policies vary according to room type. Please enter the dates of your stay and check what conditions apply to your preferred room.
Children and Extra Beds
All children are welcome.
One child under 18 years stays free of charge when using existing beds.
One child under 3 years stays free of charge in a crib.
One older child or adult is charged USD 15 per person per night in an extra bed.
The maximum number of extra beds/cribs in a room is 1.
Any type of extra bed or crib is upon request and needs to be confirmed by management.
Additional fees are not calculated automatically in the total cost and will have to be paid for separately during your stay.
Pets are not allowed.
When booking more than 9 rooms, different policies and additional supplements may apply.
Cards accepted at this property
Hover over the cards for more info.
Guests are required to show a photo ID and credit card upon check-in. Please note that all Special Requests are subject to availability and additional charges may apply.


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If we are cleaning the carpet, we might as well clean the chairs too. At this job we had over 100 red folding chairs to be cleaned, here is the last of them in line waiting to be cleaned.
If we are cleaning the carpet, we might as well clean the chairs too. At this job we had over 100 red folding chairs to be cleaned, here is the last of them in line waiting to be cleaned.
The bad part about having an area rug in the same spot for too long is that the carpet starts to get dirty and wear out everywhere except for where the rug is at. We got called to clean this home because after they moved the area rug and tried to clean it with a rug dr, you could still see where the rug used to be. The carpet will still be worn where the rug wasn't and brand new where it was, but with one pass from the truck mounted steam cleaning system, you can tell it's going to make a big difference.
Cleaned a hallway today in a commercial building that had some recent construction on the walls to make it ready for a new tenant. So bedsides the usual dirt and grime, there was a lot of dry wall parts and dust. It was no mach for Carpet Care Cleaners.
While we've talked about the things to-do during a water damage crisis, there are a few things you should never do during a flood.
Many realtors recommend that you clean the carpets before listing your home. We have worked with Brandon Nelson many times to give the homes he sells that freshly cleaned feeling, He always goes above and beyond for his customers, so we did the same. On this home we gave Brandon's client a nice little bonus after cleaning the carpet, clean tile. The entry way to this home was tiled, and although all the carpets were cleaned, it didn't seem right to leave without cleaning the entry too. Clean tile will give the best first impression possible to potential buyers.
Loved your work,
We had a very nice young man come and do our carpets. They were like new when he left. And also white again. Couldn't believe it. Fantastic job and I have recommended him to others.
Cleaning tile can be a lot of fun. People often think that because tile is a hard surface, once they mop it, it'll be clean. Dirt, grease, and grim, can sink deep into the grout lines and mopping will not make it white again. We can pressure wash the tile and grout, keep it fully enclosed, and suck everything away leaving a fresh clean space again. Sometimes it just feels like magic.
Carpet Care Cleaners did an awesome job cleaning our terribly dirty carpets! He showed up on time and was done really quickly and my carpet looks so much better. I will definitely use them again!
-Lisa Black


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Credit Suisse expands Australia equity derivatives with Kiely hire
Lucas Kiely has been appointed director and head of equity derivatives sales for Credit Suisse in Australia. Kiely will be based in Sydney. He was formerly head of equity derivatives and equity-linked sales for Australia at UBS. Before his sales role, Kiely spent six years trading structured and fund-linked derivatives across a range of asset classes both in London and Sydney. Kiely will report to Min Park, Hong Kong-based co-head of equity derivatives and convertibles, Asia-Pacific, for Credit...
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Skip to comments.Last glimpse inside ancient enigma[UK][Silbury Hill]
Posted on 01/31/2008 8:31:26 AM PST by BGHater
Silbury Hill remains an enigma despite extensive excavations
Inside Silbury Hill
You're in your jouncing people-carrier, taking in the agreeable but unremarkable view, and then suddenly it's upon you; a pointy attention-grabber at the side of the road, towering street furniture in the shape of a hazard-warning equilateral. This is crushing historical time expressed in trigonometry.
Old Egypt hands could be forgiven for thinking that the terrible shark's fin that I'm talking about is the sort of thing that looms in your windshield as you're driving through the suburbs of Cairo.
But they'd be wrong. Or they'd be half-right. Silbury Hill is a pile of chalk just off the A4 in Wiltshire but it's been called "Europe's answer to the pyramids".
There's its silhouette, for a start. Then there's the fact that the tons of calcium carbonate which went to make it were being quarried at about the same time as the pharaonic tombs were going up.
Perhaps the most pertinent similarity is that no one knows exactly how Silbury was built.
Indeed, as you round a bend out of Marlborough and come face to face with the hill, the thought that our distant kin are credited with building something so utterly alien is what immediately connects it with the ziggurats of the Nile delta.
Silbury Hill
130ft mound
completed about 2350 BC
excavated in 1776, 1849, 1968
No wonder that it's said to have been a clod of earth which fell from the Devil's foot as he flew over the Wessex plains, or a landing beacon for UFOs.
Others maintain that it's the mausoleum of a long-lost monarch. King Sil (hence 'Sil-bury') and that he's interred with a statue of his faithful steed, cast entirely from gold.
In fact, no one's sure what they got up to at Silbury as much as 4,400 years ago, though the latest dig has come out strongly in favour of singing and against sacrifices.
On a bright morning not so long ago, motorists may have seen adepts of early music ascending the 40 metre hill with their horns and their fifes, their skins and their bladders, in order to establish that sound carried from the summit to the plains below.
The hill was a bandstand, at least for some of the time. Later, under Norman rule, it was a lookout post.
There are no signifiers of ritual killings, apparently. Silbury is a very clean site, in the jargon, though not as a clean as it must have been before turf grew over the chalk, when it might have looked from a distance like a mammoth's tusk.
Such are the results of the painstaking proddings and scrapings of English Heritage, who are responsible for the hill.
Earlier prospectors weren't so kid-gloved, sinking a mineshaft into the crown and hacking into the flanks. Locals piled in, hopeful of stumbling upon King Sil's 24-carat nag.
The effects of those clumsy fumblings became apparent a few years ago when a hole opened at the apex of the hill. The tunnelers had disturbed the chalk, creating voids or air holes.
Enigmatic silence
We were the last civilians to walk inside the hill
A great English monument was in danger of falling in on itself like a sagging Yorkshire pudding.
One last excavation was sanctioned and a team from Newsnight joined it in its final stages.
The historians have uncovered arrowheads, and dense, stony antlers which were used as tools.
They now know that Silbury didn't go up all at once but in three stages. Deep inside the hill, beneath up to half a million tonnes of ever so slightly unstable chalk, we goggled at the perfectly preserved fronds of moss which had grown four millennia ago on the slopes of the original mound.
We were the last civilians to walk inside the hill, along its tunnels, beneath groaning beams that wouldn't have been out of place in a gold mine.
That's what impressionable or avaricious people thought Silbury was, of course, though the gilded gee-gee was never recovered, and the latest technology has failed to find any human remains, royal or otherwise.
English Heritage believe that the only way to conserve the hill is to close it up. Miners now labour below ground to pack the chalk and seal it with a special paste. In a few days or weeks, the grail of thousands of years will be beyond reach and Silbury - the great tepee, the big tent, of our woad-striped forefathers - will be restored to enigmatic silence.
One for you guys?
Look! A pile of dirt!
Roman clues found at ancient hill (UK).
BBC | Saturday, March 10, 2007
Posted on 03/10/2007 10:42:54 AM EST by Jedi Master Pikachu
Work Begins To Uncover Secrets Of Silbury Hill
The Telegraph (UK) | 5-12-2007 | Richard Savill
Posted on 05/12/2007 1:43:08 PM EDT by blam
In pictures: Inside Silbury Hill
BBC | Wednesday, October 24, 2007 | unattributed
Posted on 11/03/2007 1:05:42 PM EDT by SunkenCiv
related / also of interest:
Buried Megaliths Discovered At Stone Circle Site (Avebury)
Ananova | 12-2-2003
Posted on 12/02/2003 8:34:50 PM EST by blam
Stonehenge Didn't Stand Alone, Excavations Show
National Geographic | 1-12-2007 | James Owen
Posted on 01/13/2007 6:00:37 PM EST by blam
Thanks dynachrome.no one's sure what they got up to at Silbury as much as 4,400 years agoNot long after the Giza pyramids.
· Mirabilis · Texas AM Anthropology News · Yahoo Anthro & Archaeo ·
· History or Science & Nature Podcasts · Excerpt, or Link only? · cgk's list of ping lists ·
"The members of R1b are believed to be the descendants of the first modern humans who entered Europe about 35,000-40,000 years ago ( Aurignacian culture). Those R1b forebearers were the people who painted the beautiful art in the caves in Spain and France. They were the contemporaries (and perhaps exterminators) of the European Neanderthals.
BTW, I'm R1b.
12,000 Years Ago. (After the Ice Age)
Dude! It's a pile of rocks covered with dirt. Don't dis the pile!
So YOU did in the neanderthals!
Where is the endangered species act when ya need it?
Nah. All their women ran off with boys from our clan. Those are the red headed amoungst us.
I'm probably R1b too. My people have been painting themselves blue and running naked into battle since before Stonehenge was built.
Painting themselves blue and running naked, huh? Kinda reminds me of college.
My mtDNA is 'V'. A Skolt Sami of all things.
I've not been tested but with 3 of 4 grandparents with Welsh surnames, and my brown hair and green eyes, chances are pretty good I'm R1b. You don't get much more Welsh than being named Diones (Jones), Mwr (Moore), and Powys (Powers.)
Of course I do. Both my parents and all my aunts and uncles did, too.
Powis (Powys) Castle on your map is our ancestral home. Not that I could just walk in and plop down in front of the fire, mind you.
Neat. ( Atlantic Modal Haplotype)
My R1b, DYS390 is 23, which indicates a Nordic ancestry and we (probably) arrived in the Isles as one of the invading forces, Saxons, Danes or Vikings. My BIL is, R1b, DYS390-24...definately Irish.
They'd probably let me muck out the stables when I visit. If my relatives had been important, they never would have left.
I have the same 'feeling' about mine.
I looked into getting my blood tested a while ago, but can no longer find the link where I read the information... Do you by chance know the link I'm speaking of — it was to participate in some genetic study and I think I ran across the information here at FR?
Anyway, I was born with red hair which I was told fell out and then grew back in blondish as a young child and then has gotten progressively darker as time goes by, all with red highlights underneath though... Also, fair-skinned, freckled, short, and a wee bit stocky (I easily gain muscle-mass when working at it at all). My eldest daughter who looks the most like me has even more red in her hair than I. So, I'm guessing my ancestors were R1b somewhere along the line (I'm adopted, so I don't know what nationality my ancestors are/were). This stuff is always so fascinating to me.
Probably here.
"So, Im guessing my ancestors were R1b somewhere along the line (Im adopted, so I dont know what nationality my ancestors are/were). "
Red hair is not exclusive to R1b. My neighbor is haplogroup I1a and he and his brother are red haired.
Very cool! Thank you so much for the link/ping(s). :)
That is Arthur's hood.
Oh woad, woad is me!
Folks who get a chance to see it should also check out nearby Avebury.
I've been thinking about getting one of those cheek swabs...I'll freak out, though, if it unexpectedly shows French ancestry.
This aerial view of the Avebury stone circle shows the circle enclosing the village. In 2003 missing stones forming part of the great stone circle were discovered underground using computer equipment during surveys conducted by the National Trust. Avebury Circle, built 2800-2700 bc is a Bronze Age causewayed campa circular structure with an entrance at the four cardinal points. The circle of sarsen stones just inside the earth rampart encloses two further stone circles.
Firuzabad, Iran.
Whoa...methinks while we dig inside the mounds, we miss the bigger picture. What did the ancients see that gave rise to this symbolism?
What did the ancients see that gave rise to this symbolism?
That is a darn good question...
My mother's family (Colman) has a history going back to the 1700s, and their wives' families are equally interesting. The family name Williams is Welsh, I am told. We also have a strong concentration of Dutch ancestry, with some Belgian connections. I should get my brother to be tested... as I remember, it was for male descendants, wasn't it?
One of my wife's ancestors was an upper class English lady. She fell in love with one of the hired hands and they ran off to America.
Actually, this particular project is for both females and males. :) You can read more about it at the link blam shared here: http://www.freerepublic.com/focus/f-news/1423472/posts
I'm thinking of asking my husband to get tested as well. His family is Polish on both sides as far back as we have been able to trace thus far. It would be interesting to see from his DNA if there was indeed a migration or intermarriage, or if they had always lived in that area (at least as far back as scientists could tell from this! LOL).
You have the same DNA as your brother.
The only difference is that he will pass on his Y-Chromosome (male) DNA to his children but not his Mitochondral(female) (mtDNA) DNA. If you are a female, you will pass on your mtDNA(female) but not your yDNA(male) DNA to your children. You (and your brother) have your mother's mtDNA and your father's yDNA.
My father and his mother are both dead. I was able to test my dad's mothers DNA by checking his sisters daughter...my cousin. BTW, my dad's mother was a Smith and she had DNA haplogroup U5a1a. Her relatives were from Scotland when it was still possible to walk (no North Sea) and visit her relatives who are still in Finland.
Probably R1a(see post #8), maybe 'I'. See here for some info.
As above, so below?
Living in Paradise
In summary, for a thousand years during the "Age of the Gods," from about 4200 BC to 3100 BC, it was Paradise on Earth. The climate in the Middle East was mild and wet. With Earth in an orbit below Saturn, the Sun would not have been at the center of the orbit. The northern hemisphere would have faced the Sun year-round, and there would not have been the drastic changes in seasons we experience today. Mankind lived in harmony with the Gods, who supplied the techniques of agricultural cultivation, the plans for cities and temples, and the knowledge of the wheel, writing, and music. And for a thousand years the images in the sky provided a spectacular show.
"Worldwide drawings and symbols of the once-dominant luminary show a disc with rays, a disc with spokes, a disc with a central orb or eye, a disc with a crescent upon it"
It's been quite a month for this kind of thing, no?
just add water and there you have it...the legendary Atlantis, possibly constructed according to a celestial phenomenon.
I see they have a White Castle At the southern end. I love those Burgers!
Yup, it's a shame...many have begun living off their royalties.
Those boxwoods are HUGE! Must be hundreds of years old.
Very interesting! Thank you so much for the links. I find it fascinating that such diverse cultures/areas/civilizations have so much in common in ancient times. One can even draw parallels between some of the mythology and Biblical history as well. Fascinating stuff indeed!
FWIW, my family claims German but as far as I can guess is more "Prussian" as the greatest concentration of my rather rare last name is from the Grand Duchy of Mecklenburg. I have no idea where they came from before that.
When you arent fixed up for a night out do you have something of a...um...unusually youthful, almost childlike appearance?(rhetorical)
Despite middle age, without my beard, when going out I would get carded.
Somewhere along the line I read an account written by someone facing Prussian troops who described the ones he was facing as unusually stout and boyish in appearance...now I wish I knew where I had read that...
Ya'all perhaps claimin' to be related to Commander Butt Naked?
Just wonderin"
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