Nur77 protects the bladder urothelium from intracellular bacterial infection

Abstract

Intracellular infections by Gram-negative bacteria are a significant global health threat. The nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) was recently shown to sense cytosolic bacterial lipopolysaccharide (LPS). However, the potential role for Nur77 in controlling intracellular bacterial infection has not been examined. Here we show that Nur77 protects against intracellular infection in the bladder by uropathogenic Escherichia coli (UPEC), the leading cause of urinary tract infections (UTI). Nur77 deficiency in mice promotes the formation of UPEC intracellular bacterial communities (IBCs) in the cells lining the bladder lumen, leading to persistent infection in bladder tissue. Conversely, treatment with a small-molecule Nur77 agonist, cytosporone B, inhibits invasion and enhances the expulsion of UPEC from human urothelial cells in vitro, and significantly reduces UPEC IBC formation and bladder infection in mice. Our findings reveal a new role for Nur77 in control of bacterial infection and suggest that pharmacologic agonism of Nur77 function may represent a promising antibiotic-sparing therapeutic approach for UTI.

Fig. 1. Mice lacking Nur77 permit increased UPEC persistence in bladder tissue.

A Schematic of UPEC UTI mouse model. A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. B UPEC titers in urine during the first week of infection; data combined from five independent experiments. Kruskal–Wallis P < 0.0001; ***P = 0.0010 by two-tailed Mann–Whitney U test. C UPEC titers in urine collected weekly; data combined from two independent experiments. D Acute infection UPEC titers in bladder tissue homogenates; data from one experiment per time point. E Quiescent reservoir UPEC titers in bladder tissue homogenates; data from two independent experiments per time point. Central tendency lines in graphs (B, D, E) denote geometric mean values. Kruskal–Wallis P < 0.0001; *P = 0.0119, **P = 0.0035, by two-tailed Mann–Whitney U test. Source data are provided as a Source Data file.

Fig. 2. WT and Nur77-KO mice display similar patterns of immune cell populations in the bladder during and following the resolution of UPEC UTI.

A–F Bladders collected 24 h after mock or UTI89 infection. A H&E staining of formalin-fixed paraffin-embedded bladder tissue sections. Scale bar = 100 μm. B, C Comparison of the numbers (B) and relative abundances (C) of immune cell types between WT and Nur77-KO mice, combined from two independent experiments. D Gating strategy for UTI89-RFP+ immune cells. E Distribution of UPEC+ myeloid cells in WT and Nur77-KO bladders. F Percentage of each myeloid cell type that was UPEC + . G–M Bladders collected 4 weeks after mock or UTI89 infection. All infected mice in this analysis resolved bacteriuria and harbored quiescent UPEC reservoirs at this time point; data combined from two independent experiments. G H&E staining of formalin-fixed paraffin bladder tissue sections. Scale bar = 100 μm. H, I Comparison of the numbers (H) and relative abundances (I) of immune cell types between WT and Nur77-KO mice. J–M Comparison of myeloid (J, L) and lymphoid (K, M) relative abundances between uninfected and infected mice of each genotype. WT (J, K); Nur77-KO (L, M). All graphs show mean +/− SEM. J *P = 0.005256; L ***P = 0.00119, **P = 0.000985 (PMNs), P = 0.000725 (DCs), *P = 0.010793; (M) **P = 0.003107 (T cells), **P = 0.002273 (NKs) by two-tailed Mann–Whitney U test with Holm–Šídák method. Source data are provided as a Source Data file.

Fig. 3. UPEC produce more intracellular bacterial communities (IBCs) in the bladders of Nur77-KO compared to WT mice.

A Representative images of splayed bladders collected 6 hpi and stained with X-gal to visualize IBCs. IBCs were enumerated in three independent experiments (see Fig. S5 for data from third experiment). Graphs show the mean +/− SEM. 6 hpi WT and KO n = 5; 24 hpi WT n = 7, 24 hpi KO n = 6. *P = 0.0438 (6 hpi), *P = 0.0239 (24 hpi) by two-tailed Mann–Whitney. Source data are provided as a Source Data file. B Visualization of IBCs (arrows) in bladder sections collected 6 hpi. Left panels stained with H & E. Right panels stained with a primary antibody to keratin 5 (K5, green) and DAPI. UPEC-RFP appear in red. Scale bars = 100 μm.

Fig. 4. Cytosporone B inhibits UPEC intracellular infection of 5637 urothelial cell line in vitro.

A Schematic of “Concurrent Treatment” experiments in which 5637 cells were treated with DMSO vehicle control (−) or increasing concentrations of CsnB and infected with UPEC at the same time. B Time course of intracellular UPEC titers in the Concurrent Treatment model. # denotes no detectable CFU. Data are from n = 3 biological replicates per experimental condition (see Fig. S8A for additional independent experiment). ****P < 0.0001, ***P = 0.0004, **P = 0.0053 (6 hpi DMSO vs 10 μM), **P = 0.0019 (6 hpi 10 vs 100 μM), *P = 0.0240 by one-way ANOVA with Šídák’s multiple comparisons test. C Schematic of “Post-invasion Treatment” experiments in which 5637 cells were first infected with UPEC for 30 min to allow invasion to occur, and then treated with vehicle or CsnB. D Time course of intracellular UPEC titers in the Post-invasion Treatment model. Each experimental condition includes n = 6 biological replicates from two independent experiments (each with n = 3 biological replicates). **P = 0.0026 (4 hpi DMSO vs 100 μM), **P = 0.0017 (4 hpi 10 vs 100 μM), *P = 0.0232 (2 hpi DMSO vs 100 μM), *P = 0.0494 (2 hpi 10 vs 100 μM), by one-way ANOVA with Šídák’s multiple comparisons test. Graphs in (B, D) show the mean with SEM. A, C Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Fig. 5. CsnB induces UPEC expulsion from 5637 urothelial cell line.

A Schematic of “Concurrent Treatment” experiments in which 5637 cells were treated with DMSO vehicle control (−) or increasing concentrations of CsnB and infected with UPEC at the same time. UPEC CFU were enumerated in the supernatant and expulsion was calculated relative to the initial load of intracellular UPEC enumerated in parallel wells that were lysed after the 30 min invasion period. B Time course of UPEC expulsion from cells treated concurrently with CsnB. # denotes no detectable CFU. Data are from n = 3 biological replicates per experimental condition. C Schematic of “Post-invasion Treatment” experiments in which 5637 cells were treated with DMSO vehicle control (−) or increasing concentrations of CsnB only after the 30 min invasion period. D Time course of UPEC expulsion from cells in the post-invasion treatment model. Each experimental condition includes n = 6 biological replicates from two independent experiments (each with n = 3 biological replicates). Both graphs denote the mean with SEM. *P = 0.0146, ***P = 0.0003, by one-way ANOVA with Šídák’s multiple comparisons test. Source data are provided as a Source Data file. A, C Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Fig. 6. Cytosporone B inhibits UPEC invasion and blocks endocytosis in 5637 urothelial cell line in vitro.

A Schematic of 5637 cell invasion assay. A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. B Intracellular UPEC titers 30 min post infection in cells treated concurrently with DMSO vehicle control (−) or increasing concentrations of CsnB. ****P < 0.0001, **P = 0.0046 (DMSO vs 10 μM), **P = 0.0050 (1 vs. 10 μM) by one-way ANOVA with Šídák’s multiple comparisons test. C Relative UPEC invasion 30 min post inoculation, calculated from the CFU data in (B). Each experimental condition in (B, C) includes six biological replicates from two independent experiments. **P = 0.0019 (DMSO vs 100 μM), **P = 0.0014 (1 vs 100 μM) by Kruskal–Wallis with Dunn’s multiple comparisons test. D Histograms of a time course of intracellular transferrin detected by flow cytometry. The dotted line denotes gating for defining transferrin + cells in (E). E Time course of transferrin positivity expressed as % of live cells in each condition. ****P < 0.0001, **P = 0.0065 by one-way ANOVA with Šídák’s multiple comparisons test. D, E Are representative to two independent experiments, each with three biological replicates for each condition and time point. Bars denote the mean with SEM. Source data are provided as a Source Data file.

Fig. 7. Mice treated with CsnB are protected from UPEC bladder infection.

A Schematic of CsnB treatment and UPEC UTI mouse model for data in (B–D). B Enumeration of IBCs in splayed bladders 6 hpi. n = 10 mice per group. Results are combined from two independent experiments. *P = 0.0198 by Mann–Whitney test. C Time course of UPEC titers in urine. **P = 0.0016 by Kruskal–Wallis with Dunn’s multiple comparisons test. D UPEC titers in bladder and kidney tissue homogenates 1 wpi. **P = 0.0036 by Mann–Whitney test. C, D DMSO-treated n = 7, CsnB-treated n = 8. E Schematic of CsnB treatment and UPEC UTI mouse model in WT (DMSO-treated n = 10, CsnB-treated n = 9) and Nur77-KO (n = 6 per group) mice for data in (F). F UPEC titers in bladder tissue homogenates. Results are combined from two independent experiments. ***P = 0.0007 by Mann–Whitney test. Graphs in (C, D, F) denote geometric mean values. Graph in B shows mean +/− SEM. Source data are provided as a Source Data file. A, E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.