Fig 1.
Acute cystitis strains activate an IL-1β response in human bladder epithelial cells.
(A) IL-1β response in human bladder epithelial carcinoma cells (HTB-9) infected with acute cystitis strains CY-92, CY-17, CY-132 and CY-49 (4 hours). CFT073 and E. coli 83972 (ABU) were used as reference strains. CY-17, CY-92 and CY-132 triggered high IL-1β responses, as did CFT073. IL-1β was quantified, in cell supernatants, by ELISA (means ± SEMs of three independent experiments, *** P < 0.001 compared to PBS, two-tailed unpaired t-test). (B) Lack of IL-1β secretion by infected human kidney epithelial carcinoma cells (A498), infected as in Fig 1A. (C) Increased pro-IL-1β and mature IL-1β levels in HTB-9 cells infected with CY-92, CY-17 and CY-132 (Western blot analysis of cell supernatants, 4 hours, one representative experiment of several repeats). (D) IL-1β staining of HTB-9 cells infected with CY-17, CY-92, CY-132, CFT073 or ABU compared to the background in uninfected cells (PBS). Scale bars = 20 μm (upper panel) and 10 μm (lower panel). One representative experiment is shown. (E) Quantification of the cellular IL-1β response to infection. Increase in total fluorescence intensity (open pin-hole) after subtraction of the background staining in uninfected cells (PBS) (means ± SEMs of 50 cells per sample, ** P < 0.01 and *** P < 0.001 compared to PBS, two-tailed unpaired t-test). One of three experiments is shown. (F, G) IL-1β response to infection (1 hour) quantified by Western blot of whole cell extracts. Quantification of integrated density relative to GAPDH normalized against the background of uninfected cells. One representative experiment of several repeats. (H) IL-1β response to an epidemiologically defined collection of pediatric acute cystitis strains (n = 67) compared to ABU strains (n = 62), obtained from children in the same geographic area. IL-1β was quantified in infected cell supernatants, by ELISA. Pie chart depicting the frequency of bacterial strains activating IL-1β responses: high (orange), intermediate (blue), low (purple) or negative (green). Histogram (inset) of the mean IL-1β response to CY versus ABU strains (means ± SEMs, *** P < 0.001, two-tailed Mann Whitney test). (I) IL-1β activation plotted against hemolytic activity in the collection of CY and ABU strains. No significant association was detected (n = 18–21, Hly+ versus Hly-, two-tailed Mann Whitney test).
Fig 2.
Genetic control of inflammation in acute cystitis.
(A) Macroscopic evidence of acute cystitis in CFT073 infected mice, 7 days after infection. Slight increase in edema and hyperemia in C57BL/6 WT mice compared to uninfected controls, massively enlarged, hyper-inflamed bladders in Asc-/- and Nlrp3-/- mice and intact morphology in bladders from Il1b-/- mice. The macroscopic pathology score was based on edema, hyperemia and size on a scale of 0–10, where 10 the most edematous, hyperemic and largest bladder). Individual pathology scores are indicated. Scale bar = 1 mm. Two representative bladders are shown for each genotype. (For uninfected Asc-/-, Nlrp3-/- and Il1b-/- mice see S2A Fig). (B) Bladder tissue structure in H&E-stained tissue sections. Bladders from Asc-/- and Nlrp3-/- mice showed extensive edema, a loss of tissue structure and epithelial hypertrophy, compared to C57BL/6 WT and Il1b-/- mice or uninfected control mice. The histology score was assessed using H&E stained bladder sections based on neutrophil infiltration, tissue architecture and epithelial thickness on a scale of 0–10, where 10 is highest neutrophil infiltration, least preserved tissue architecture and maximum epithelial thickness). Individual histology scores are indicated. Scale bar = 200 μm. (C) Gross bladder pathology score in infected mice (7 days) and uninfected C57BL/6 WT controls (means ± SEMs of two experiments, *** P < 0.001, two-tailed unpaired t-test compared to WT mice). (D) Elevated neutrophil counts and (E) bacterial counts in urine and (F) bladder tissues from Asc-/- and Nlrp3-/- mice, compared to WT and Il1b-/- mice. Means ± SEMs of two experiments, *** P < 0.001, ** P < 0.01, * P < 0.05, two-tailed unpaired t-test compared to WT mice. (G) Detection of neutrophils and bacteria in the mucosa of infected and control mice, by immunohistochemistry. Increased staining for mucosal neutrophils (red) and bacteria (green) in Asc-/- and Nlrp3-/- mice, compared to C57BL/6 WT, Il1b-/- mice and uninfected controls. Tissues obtained 7 days after infection. Scale bar = 50 μm. (H) Mucosal IL-1β staining in bladder tissue sections, obtained 24 hours after infection. Infection increased epithelial IL-1β staining in C57BL/6 WT, Asc-/- and Nlrp3-/- mice. Scale bar = 50 μm. Experiments included 14 Asc-/-, 11 Nlrp3-/-, 11 C57BL/6 WT and 10 Il1b-/- mice.
Fig 3.
Hyper-activation of IL-1β dependent gene expression and bladder pathology in Asc-/- and Nlrp3-/- mice.
Transcriptomic analysis of whole bladder RNA from infected mice (CFT073, 7 days), compared to uninfected controls of each genotype (cut off FC 1.41, P < 0.05). (A) Heatmap of regulated genes in Asc-/- and Nlrp3-/- mice with the highest bladder pathology score, defined by neutrophil infiltration, loss of tissue structure and epithelial thickness in H&E stained bladder tissue sections. Scale FC -4 to 4, red = upregulated, blue = downregulated. A distinct gene set distinguished the Asc-/- and Nlrp3-/- mice with a high histopathology score from C57BL/6 WT mice or Il1b-/- mice without pathology. (B) Top up-regulated genes in the pathology-associated gene set, compared to uninfected controls of each genotype. Means ± SEMs of 5 mice for Asc-/- mice and 2 Nlrp3-/- mice. (C) Analysis of IL-1β, inflammasome activators and effectors in Asc-/- and Nlrp3-/- mice, detecting massive over-expression compared to Il1b-/- and WT mice. Red = upregulated, blue = suppressed. The data set included gene expression profiles from 7 Asc-/- and 5 Nlrp3-/- mice, and two each of the C57BL/6 WT and Il1b-/- controls. Uninfected control RNA of each genotype were used to define significantly regulated genes (≥ 2 mice per genotype). Histopathology scores and group numbers for individual mice (see also Experiments 1, 2 and 3 in S1 Table).
Fig 4.
(A) Gene expression profiling identified Mmp7 as one of the top up-regulated gene in Asc-/- and Nlrp3-/- mice with bladder pathology (CFT073 infected mice, 7 days). Log2 fold change of Mmp7 expression levels in individual mice are shown relative to the H&E pathology score. Mmp7 was not regulated in C57BL/6 WT mice or in Il1b-/- or Casp1-/- mice. (B) Strong epithelial MMP-7 staining in Asc-/- and Nlrp3-/- mice with bladder pathology. MMP-7 staining was very low in C57BL/6 WT, Il1b-/- and Casp1-/- mice. Scale bars = 50 μm. (C) Phenotype of Mmp7-/- mice, 7 days after infection with CFT073. Intact mucosal tissue structure with inflammatory cell infiltration. Low bacterial and neutrophil counts in urine compared to Asc-/- mice (n = 5 mice per group, means ± SEMs, ** P < 0.01, *** P < 0.001, two-tailed unpaired t-test). Scale bar = 1 mm. IL-1β levels were elevated in the urine of Asc-/- mice but not in Mmp7-/- mice, as detected by ELISA. (D) Proteolytic cleavage of pro-IL-1β by MMP-7 in vitro, using purified enzyme and GST-tagged pro-IL-1β. The IL-1β fragments generated by proteolysis were 18 and 16 kDa, defined by Western blot using an antibody specific for the mature form of IL-1β. Recombinant mature IL-1β and GST-tagged pro-IL-1β were used as controls, as well as recombinant MMP-7. One representative experiment out of three, see also S7A Fig. (E) Bioassay for IL-1β activity, measuring the PGE2 response of human bladder epithelial cells to the IL-1β fragments generated by MMP-7 proteolysis of GST-tagged pro-IL-1β. The cleaved products activated PGE2 but MMP-7 and pro-IL-1β had no effect (means ± SEMs of two experiments, ** P < 0.01, two-tailed Mann Whitney test).
Fig 5.
Regulation of MMP7 expression by ASC and NLRP-3.
(A) MMP-7, ASC and NLRP-3 responses to infection were visualized by confocal microscopy. An increase in MMP-7 and decrease in ASC staining were detected after infection of HTB-9 cells with CY-17 and CY-92 for 1 hour, compared to uninfected control cells. NLRP-3 staining was weakly affected. (B) Quantification of total fluorescence intensity (open pin-hole) after subtraction of the background staining in uninfected cells (PBS). Medians ± SEMs of 50 cells, * P < 0.05, ** P < 0.01, *** P < 0.001, compared to PBS control, two-tailed unpaired t-test (MMP-7 and NLRP-3) or two-tailed Mann Whitney test (ASC). One of three experiments is shown. (C) Western blot confirming the change in cellular content of MMP-7, ASC and NLRP-3, 1 hour after infection with the indicated strains. Fold change compared to PBS of normalized values (against GAPDH). One experiment out of 2 is shown. (D) Increase in MMP-7 expression in HTB-9 cells transfected with siRNAs specific for ASC or NLRP3 and infected with CY-17 (4 hours, scale bars = 20 μm). (E) Western blot confirming the knock-down of ASC or NLRP-3 with siRNAs. A further reduction in ASC expression was detected after CY-17 infection (4 hours, quantified in S8B Fig, one experiment out of 2 is shown.). (F) PCR amplification of a 259 bp fragment in the MMP7 promoter (P1, -18/-276 relative to the transcription start site). (G) EMSA of the amplified fragment and nuclear extract from CY-17 infected HTB-9 cells (4 hours). Binding of ASC and NLRP-3 to P1 was identified as a band shift (arrow indicating protein-DNA complex). The band shift was inhibited by ASC- or NLRP-3-specific antibody. Free DNA formed a single low molecular weight band (arrow indicating free probe). The band shift was not affected by the IgG isotype control. One of three similar experiments is shown. (H) EMSA of the 259 bp MMP7 promoter fragment P1 and recombinant ASC. Dose-dependent formation of an ASC-P1 complex is shown as a band shift (arrow indicating ASC-DNA complex), which was inhibited by 0.5 and 1.0 μg of anti-ASC antibodies. The band shift was not affected by negative control murine IgG control. One of three similar experiments is shown.
Fig 6.
Acute cystitis immunotherapy, using an IL-1 receptor antagonist (IL-1RA) or an MMP inhibitor.
(A) Overview of therapeutic regimen used to inhibit bladder pathology. Asc-/- mice were pre-treated with Anakinra (IL-1RA), 30 min before infection and daily after infection with E. coli CFT073 (1 mg in 100 μl of PBS i.p. per mouse) and sacrificed 7 days after infection. Alternatively, Asc-/- mice were pre-treated with the matrix metalloproteinase inhibitor (MMPI) Batimastat, 30 min before infection and daily after infection with E. coli CFT073 (0.5 mg in 100 μl of PBS i.p. per mouse, except day 3) (n = 7 per treatment group, total of two experiments). (B) Difference in gross bladder pathology between untreated controls and IL-1RA or MMPI treated mice. Two representative mice per group are shown. The IL-1RA therapy reduced macroscopic bladder pathology in Asc-/- mice. Scale bar = 1 mm. The MMPI therapy showed a similar but less pronounced effect. (C) Edema, hyperemia and size of the bladders were used as scoring parameters for the pathology score. Pathology scores from individual mice are shown. The gross pathology score was reduced by the inhibitors (P < 0.001, for IL-1RA compared to untreated Asc-/- mice and P = 0.002, for MMPI, compared to untreated Asc-/- mice, means ± SEMs of two experiments, two-tailed Mann Whitney test). (D) Protection from bladder tissue pathology shown in H&E stained sections from treated versus control mice. Arrows indicate mucosal sloughing, edema and subepithelial abscesses in untreated mice. Inhibition of mucosal neutrophil aggregate formation in bladder sections from treated mice compared to untreated and infected mice. Scale bar = 200 μm (H&E) and 50 μm (immunofluorescence). (E) Kinetics of neutrophil recruitment and bacterial clearance in the urine of IL-1RA or MMPI treated Asc-/- mice, compared to untreated mice. (n = 7 mice per group, means ± SEMs, * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed unpaired t-test).
Fig 7.
Elevated concentrations of IL-1β and MMP-7 in the urine of patients with acute cystitis.
(A) IL-1β concentrations in urine samples from patients with acute cystitis (n = 9). (B) IL-1β concentrations in consecutive urine samples from patients with ABU, who were long-term asymptomatic carriers of E. coli 83972 [21] (means ± SEMs, 20 patients, 161 urine samples). Elevated levels of IL-1β in the cystitis patients compared to the ABU group. Pie chart (inset) depicts the distribution of IL-1β concentrations in each patient group. (C) Histogram compares IL-1β concentrations between the two patient groups (means ± SEMs, *** P < 0.001, two-tailed Mann Whitney test). (D) MMP-7 concentrations were higher in urine samples from the patients with acute cystitis than in patients with long-term ABU (means ± SEMs, *** P < 0.001, two-tailed unpaired t-test). Urine samples were obtained from patients with sporadic acute cystitis at the time of diagnosis. The patients with ABU participated in a prospective study of therapeutic inoculation with E. coli 83972 and were subjected to long-term follow up [21]. Multiple samples were obtained during asymptomatic carriage (3–14 samples per patient).
Fig 8.
Models of IL-1β processing—cellular determinants and biological effects.
(A) Caspase-1 dependent pro-IL-1β processing by the NLRP-3 inflammasome in mice with intact inflammasome function. The NLRP-3/ASC complex activates Caspase-1, which in turn cleaves pro-IL-1β and supports the secretion of mature IL-1β by infected cells. The production of MMP-7 is normally low, due to transcriptional repression by ASC and NLRP-3, bound to the MMP-7 promoter. (B) C57BL/6 WT mice develop a mild form of acute cystitis with transient inflammation. Il1b-/- mice were protected against infection and inflammation and Casp1-/- mice, showed an atypical phenotype without tissue damaging inflammation or neutrophil recruitment into the urine. (C) A new mechanism of IL-1β processing by MMP-7 in infected Asc-/- and Nlrp3-/- mice. MMP-7 over-activation in Asc-/- mice and Nlrp3-/- mice is triggered by infection and de-repression of Mmp7 transcription. (D) In the absence of functional Asc or Nlrp3 genes, infected mice therefore produce excessive amounts of IL-1β, causing massive bladder inflammation, with elevated neutrophil counts, edema and hypertrophy of the bladder epithelium. (E) Immunotherapy by suppression of the IL-1-dependent inflammatory response in susceptible Asc-/- mice. Mice were treated with the IL-1RA (Anakinra) or the protease inhibitor MMPI (Batimastat), which showed therapeutic effects.