Recurrent infections drive persistent bladder dysfunction and pain via sensory nerve sprouting and mast cell activity

Abstract

Urinary tract infections (UTIs) account for almost 25% of infections in women. Many are recurrent (rUTI), with patients frequently experiencing chronic pelvic pain and urinary frequency despite clearance of bacteriuria after antibiotics. To elucidate the basis for these bacteria-independent bladder symptoms, we examined the bladders of patients with rUTI. We noticed a notable increase in neuropeptide content in the lamina propria and indications of enhanced nociceptive activity. In mice subjected to rUTI, we observed sensory nerve sprouting that was associated with nerve growth factor (NGF) produced by recruited monocytes and tissue-resident mast cells. Treatment of rUTI mice with an NGF-neutralizing antibody prevented sprouting and alleviated pelvic sensitivity, whereas instillation of native NGF into naïve mice bladders mimicked nerve sprouting and pain behavior. Nerve activation, pain, and urinary frequency were each linked to the presence of proximal mast cells, because mast cell deficiency or treatment with antagonists against receptors of several direct or indirect mast cell products was each effective therapeutically. Thus, our findings suggest that NGF-driven sensory sprouting in the bladder coupled with chronic mast cell activation represents an underlying mechanism driving bacteria-independent pain and voiding defects experienced by patients with rUTI.

Figure 1:. rUTI Induces Sensory Sprouting in the Bladder Evoking Pelvic Pain-like Behavior and Void Defects

(A-B) Bladder biopsies were obtained from (A) control and (B) rUTI patients and immunostained for E-cadherin to identify intermediate epithelium and substance P (SP) to identify the neuropeptide. Scale bar, 50 μm (C) Quantification of SP immunostain from patient bladder biopsies, with each data point representing a single field of view (n=3 control and n=8 rUTI patients). (D) Urine from control and rUTI patients was collected and assayed for SP level (n=12 control and n=25 rUTI patients). (E) Schematic of rUTI model. Mice were infected with 10⁸ CFU UPEC once a week for three consecutive weeks. Functional assays were performed 14 days after the third infection. (F) rUTI or control (saline instilled) mice were mechanically probed to assess response frequency to pelvic stimulation(n=13–17 mice). (G) Frequency was assessed via cystometry analysis of control and rUTI mice (n=7–9 mice). Data is representative of 2–3 independent experiments. (H-K) Representative images (H,J) and quantification (I,K) of fluorescence staining in the bladder show increased presence of nerve fibers (Green, TUJ1⁺) in the lamina propria but not in the detrusor in mice following rUTI. Each plot point represents one image field. Data is representative of 2–3 independent experiments, with each containing at least two mice per treatment group. Scale bar, 100 μm. (L-N) (L) Representative (left) immunofluorescence and (right) 3D models and (M,N) quantification of SP⁺ innervation in control and rUTI lamina propria. Both neurite length and number of branch points increase in rUTI mice compared to the controls. Z-stacks were acquired per mouse bladder from at least two independent experiments, with at least two mice per treatment group. Scale bar, 30 μm. All comparisons were analyzed by unpaired Mann-Whitney U Test with p<0.05 used to define significance. Mean±SEM, ****p<0.0001, **p<0.01, *p<0.05, ns=not significant.

Figure 2:. NGF Mediates Sensory Nerve Growth and Development of Pelvic Sensitivity during rUTI

(A,B) Bladders were harvested from control and rUTI mice 7 days after the third instillation, homogenized, and analyzed for (A) NGF and (B) BDNF protein by ELISA (n=3–6 mice). (C-E) (C) Representative (left) immunofluorescence and (right) 3D models and quantification of SP⁺ (D) neurite length and (E) number of branch points in WT bladders after daily instillation of NGF into the bladder by transurethral catheterization (n=3 mice). Scale bar, 80 μm. (F) Mice instilled with NGF daily for 7 days were assessed for pelvic sensitivity 2 days after the final instillation (n=3 mice). (G-H) Neurons from L6 and S1 DRGs were harvested from mice and treated with either vehicle, NGF (25 ng/ml) or NGF and GW-441756 (7.25 μM) overnight. Scale bar, 50 μm. (H) The percent of neurons with neurites were quantified per group (n=5–6 images, approximately 150 neurons were counted per group). (I-L) rUTI mice were treated with vehicle or GW-441756 daily (IP and intravesicular instillation) starting at the second infection. Bladders were harvested 14 days after final infection and probed for SP to assess innervation. (I-K) (I) Representative (left) immunofluorescence and (right) 3D models and quantification of SP⁺ (J) neurite length and (K) number of branch points in rUTI mice treated with either vehicle or GW-441756 (n=5–6 mice per group (L) A separate group of vehicle or GW-441756 treated rUTI mice were assessed for pelvic sensitivity 14 days after final infection (n=4–6 mice). Z-stacks were acquired per mouse bladder from at least two independent experiments, with at least two mice per treatment group. Scale bar, 70 μm. (M-O) (M) Representative (top) immunofluorescence and (bottom) 3D models and quantification of SP⁺ (N) neurite length and (O) number of branch points in mice treated weekly (intraperitoneal injection; IP) with a NGF neutralizing antibody during rUTI (n=5 mice per group). Scale bar, 80 μm. (P) Mice undergoing NGF neutralizing were also assessed for pelvic sensitivity 7 days after the final anti-NGF treatment (n=5 mice per group). (A,B,D-F,J-L) Comparisons were analyzed by unpaired Mann-Whitney U Test with p<0.05 used to define significance. (H,N-P) Comparisons were analyzed by one-way Anova with Tukey post hoc test. p<0.05 used to define significance. Mean±SEM, ****p<0.0001, **p<0.01, *p<0.05, ns=not significant.

Figure 3:. Infiltrating monocytes and resident mast cells mediate sensory sprouting via NGF production after rUTI

(A-B). Flow cytometry analysis of single-cell suspensions of bladder cells 7 days third UTI or saline to assess NGF. Corresponding gating strategy is provided in Supplemental Figure S3. Live cells were gated on NGF expression and quantifications of (A) CD45⁺ and (B) CD45^- cells were determined (n=5 mice). (C) Donut charts generated from flow cytometry analysis (see Figure S3) of CD45⁺ bladder cells at baseline, 24 hours after first UTI, 7 days after first UTI, and 24 hours after second UTI (given 7 days after first UTI). (D-F) Analysis of NGF MFI expression in (D) macrophages, (E) monocytes, and (F) mast cells (n=5 mice). (G,H) Neurons from L6 and S1 DRGs were harvested from mice and treated with either vehicle, native NGF (25 ng/ml), or cultured with bone marrow derived (G) monocytes or (H) mast cells in absence or presence of TrkA antagonist, GW-441756 (7.25 uM) overnight. The percent of neurons with neurites were quantified per group (n=6–10 images, approximately 180 neurons were counted per group). (I,J) Quantification of SP⁺ (I) neurite length and (J) number of branch points in Ccr2^−/− bladders after rUTI. (n=5 mice). (K-M) (K) Representative (left) immunofluorescence and (right) 3D models and quantification of SP⁺ (L) neurite length and (M) number of branch points in mice treated twice weekly (intraperitoneal injection; IP) with an antibody targeting Ly6C during rUTI (n=5 mice per group). Scale bar, 80 μm. (N,O) Quantification of SP⁺ (N) neurite length and (O) number of branch points in Kit^w-sh/w-sh bladders after rUTI (n=5 mice). (P-S) (P,R) Void frequency and (Q,S) pelvic sensitivity were assessed in (P) Ccr2^−/−, (Q) anti-Ly6C treated, and (R,S) Kit^w-sh/w-sh mice following rUTI (n=4–5 mice). All data representative of 2–3 independent experiments. (A-B,I,J,N-P,R,S) Comparisons were analyzed by unpaired Mann-Whitney U Test with p<0.05 used to define significance. (D-H,L,M,Q) Comparisons were analyzed by one-way Anova with Tukey post hoc test. p<0.05 used to define significance. Mean±SEM, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns=not significant.

Figure 4:. Mast Cells Mediate Pain-like Behavior and Void Defects via Mediator Release

(A) rUTI mice were treated with either vehicle or GW-441756 on days 13 and 14 after final infection and assessed for pelvic sensitivity (n=5 mice). (B,C) Bladders were harvested from control and rUTI mice and immunofluorescence was used to identify lamina propria mast cells and activated (pERK⁺) SP⁺ nerves in frozen sections. (C) Mast cells (identified by avidin staining; pointed by the arrow) were noted in proximity of sensory nerves. Scale bar, 50 μm. (B) Increased presence of activated nerves (pERK⁺SP⁺) in rUTI bladders compared to saline control (n=6 mice). (D,E) (D) Control and rUTI mouse bladders were injected with fluorescent WGA (WGA-647) and L6 and S1 DRGs were harvested 5 days later to assess bladder innervating neurons. DRGs were subsequently stained with (green) Beta III Tubulin and (red) pERK as shown in the merged image. Double positive neurons are demarcated by a star (n=3 mice). Scale bar, 50 μm. (E) Quantification of pERK⁺ neurons among the traced neurons. (F) Bladders were harvested from control and rUTI Kit^w-sh/w-sh mice and activated SP⁺ nerves were quantified (n=6 mice). (G) rUTI was performed in MCPT5-iDTR mice or littermate controls prior to DT injection to deplete mast cells. Pelvic sensitivity was assessed in both groups of mice after DT injection (n=3 mice). (H) Mast cells were cultured (64) and treated with 200 μM SP for 24 hours. Supernatant was collected from untreated and stimulated mast cells and assessed for NGF protein content. (I) Urine was collected from WT control and rUTI mice 7 days after the first and second infection and 14 days after the third infection and assess for histamine levels by ELISA (n=4–11 mice). (J,K) Quantification of SP⁺ (J) neurite length and (K) number of branch points in Trpv1^−/− mice during rUTI (n=4 mice per group). (L) Trpv1^−/− mice were also assessed for pelvic sensitivity after rUTI (n=4 mice per group). (M,N) WT mice instilled with saline, bradykinin, or histamine for 15 min were assessed for (M) pelvic sensitivity and (N) frequency 1 hour after instillation (n=5 mice). (O) Trpv1^−/− mice instilled with histamine for 15 min were assessed for pelvic sensitivity 1 hour after instillation (n=5 mice). (P,Q) WT mice after control or rUTI treated with (P) pyrilamine maleate or (Q) icatibant were assessed for pelvic sensitivity (n=5 mice). (A,C,E-H,O-Q) Comparisons were analyzed by Mann-Whitney U Test with p<0.05 used to define significance. (I,M,N) Comparisons were analyzed by unpaired Student T-Test with p<0.05 used to define significance. (J-L) Comparisons were analyzed by one-way Anova with Tukey post hoc test. p<0.05 used to define significance. Mean±SEM, ***p<0.001, **p<0.01, *p<0.05, ns=not significant.