Figure 7. UPIIIa mediates FimH-induced urothelial apoptosis.

FimH-induced caspase 3/7 activation requires UPIIIa (A). Caspase 3/7 was measured in culture extracts by cleavage of fluorogenic substrate. Induction of caspase 3/7 activity by NU14 (MOI 500) was significantly inhibited in PD07siUPIII cultures (*p<0.05) compared to control, and no induction of caspase 3/7 was detected in response to NU14-1. (B) PD07siTNFR2 cells or PD07siUPIII cells were treated with 10 µg/ml FimCH for 4 h. Apoptosis was visualized using fluorescent annexin V staining (green) and propidium iodide (red) staining. Annexin staining was quantified by manual counting of fluorescently labeled cells relative to brightfield images (see Methods). Annexin-positive cells were significantly reduced in siUPIII cells relative to siTNFR2 cells (p<0.05). (C) In experiments in the presence of inhibitors, PD07i cells treated with 10 µg/ml FimCH showed reduced annexin staining when treated with the CK2 inhibitor TBB compared to DMSO (p<0.05). (D) Organotypic raft cultures of PD07i or PD07siUPIII cells were incubated with NU14 or NU14-1 for 4 h at an MOI of 100. After processing, frozen sections were stained for TUNEL (green) and counterstained with DAPI (blue). Prominent TUNEL staining was induced by NU14 only in PD07i tissue. Images are representative of staining patterns obtained from TUNEL assay of four different raft cultures. (E) UPEC-induced apoptosis was also assessed in bladder sections using TUNEL staining (green). In in vivo experiments female C57BL/6 mice were infected with 10⁸ NU14 or NU14-1 for 6 hours in the presence of DMSO (i) or 10 µM TBB (ii). Bladder sections from DMSO treated animals exhibited foci of apoptosis (arrows) that were reduced or absent in TBB-treated animals. In H&E stained bladder sections from DMSO (iii) and TBB (iv) treated mice, infiltration of large numbers of inflammatory cells (white arrows) were observed in response to infection with 10⁸ NU14 for 2 hours, suggesting an intact innate immune response. Images are of representative sections and scale bars represent 50 µm. All experiments were repeated two or more times, and statistical significance is indicated at *p<0.05 and data are represented as mean±SEM.