Research Article


  • Hans Fischer,

    Affiliation: Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden

  • Nataliya Lutay,


  • Bryndís Ragnarsdóttir,


  • Manisha Yadav,


  • Klas Jönsson,

    Affiliation: Singapore Immunology Network (SIgN), Biomedical Sciences Institutes, Agency for Science, Technology, and Research (A*STAR), Immunos, BIOPOLIS, Singapore, Singapore

  • Alexander Urbano,


  • Ahmed Al Hadad,


  • Sebastian Rämisch,


  • Petter Storm,


  • Ulrich Dobrindt,

    Affiliation: Institute for Molecular Biology of Infectious Diseases, Julius-Maximilians-University Würzburg, Würzburg, Germany

  • Ellaine Salvador,


  • Diana Karpman,

    Affiliation: Department of Pediatrics, Clinical Sciences Lund, Lund University, and Lund University Hospital, Lund, Sweden

  • Ulf Jodal,

    Affiliation: Pediatric-Uronephrology Center, Queen Silvia Children's Hospital, University of Gothenburg, Sweden

  • Catharina Svanborg mail

    Affiliations: Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden, Singapore Immunology Network (SIgN), Biomedical Sciences Institutes, Agency for Science, Technology, and Research (A*STAR), Immunos, BIOPOLIS, Singapore, Singapore

  • Published: September 23, 2010
  • DOI: 10.1371/journal.ppat.1001109



Author Summary









Increased susceptibility to acute pyelonephritis in Irf3−/− mice



Figure 1. Increased mortality and tissue damage in Irf3−/− mice after experimental UTI.

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).





Ceramide/TLR4 interactions and TRAM phosphorylation



Figure 2. Ceramide-TLR4 interactions, adaptors and TRAM phosphorylation.

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).




Kinase phosphorylation downstream of ceramide/TLR4



Figure 3. Protein phosphorylation and transcriptomic response to r-ceramide or LPS+sCD14.

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.



Transcriptional activation in response to ceramide


IRF3 translocation to the nucleus in response to ceramide/TLR4



Figure 4. Nuclear IRF3 translocation in response to ceramide/TLR4.

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).






Bacterial fimbriae and IRF3 translocation in infected human renal tubular epithelial cells



Figure 5. Nuclear IRF3 translocation after stimulation with P-fimbriated E. coli and the importance of IFN signaling for host resistance against UTI.

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 promoter polymorphisms in patients with urinary tract infection




Figure 6. Human IRF3 promoter sequence variation and activity in UTI-prone patients.

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 acute pyelonephritis-associated promoter SNPs reduced transcription efficiency












Ethics statement





Cell cultures




siRNA transfection


RT-PCR array


Phospho-kinase array


SDS-PAGE and immunoblotting


Native PAGE and immunoblotting


DNA microarray analysis



Bacteria and growth conditions


Experimental urinary tract infection (UTI)


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.


UTI-prone patients and pyrosequencing


Transient transfection and dual luciferase reporter assay





List of ID numbers for genes and proteins of mouse and humans


Supporting Information

Figure S1.



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Figure S2.



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Figure S5.



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Figure S6.



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Figure S9.



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Figure S10.



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Figure S11.



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Supporting Information S1.

Tables S1 to S5.


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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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