Fig 2: 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 3: 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.

Fig 4: IL-1β processing by MMP-7.(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: Hyper-activation of IL-1ß 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-1ß, 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).