Loss of the Sympathetic Signal Produces Sterile Inflammation of the Prostate

Abstract

Neural innervations exert essential roles in the prostate. However, spatial distribution and regulatory function of such neural inputs are incompletely characterized. Here, we exploited the advanced whole-tissue immunolabeling and optical clearing technique to assess the 3D anatomy of autonomic innervations in the mouse and human prostate for the first time. We observed that sympathetic and parasympathetic inputs in the mouse prostate remained unaffected during castration-induced tissue regression. However, the pharmacologic destruction of sympathetic innervations in the mouse prostate led to sterile inflammation of the tissue, mimicking the disease condition of chronic non-bacterial prostatitis. Also, the genetic ablation of sympathetic inputs produced a similar inflammatory response. Furthermore, we showed that treatment of the specific β2-adrenergic receptor agonists could effectively mitigate the prostate inflammation caused by such sympathetic loss. Together, these results have elucidated the new immunomodulatory function of the sympathetic signal via the β2-adrenergic receptor in prostate inflammatory disease.

Keywords: 3D fluorescence imaging; chronic non-bacterial prostatitis; prostate; sympathetic innervations; β2-adrenergic receptor.

FIGURE 1

Whole-tissue 3D fluorescence imaging of the mouse prostate. (A–C) 3D fluorescence imaging of neural innervations in the mouse prostate. (A) The intact prostate of the 8-week-old male mouse before (left panel) and after (right panel) the whole-tissue immunolabeling and optical clearing. Anterior prostate (AP) and ventral prostate lobes (VP) are indicated. (B,C) The intact prostate of the 8-week-old male mouse was subjected to the whole-tissue PGP9.5-immunolabeling. (B) Representative 3D projection of the lightsheet imaging is shown. (C) Representative optical sections (upper panels) and 3D projections (lower panels) of the 1-mm thickness of the anterior (AP) and ventral prostate (VP) of the lightsheet imaging are shown. (D,E) 3D assessment of blood vessels in the mouse prostate. The intact prostate of the 8-week-old male mouse was subjected to the whole-tissue PECAM1-immunolabeling. (D) Representative 3D projection of the lightsheet imaging is shown. (E) Representative optical sections (upper panels) and 3D projections (lower panels) of the 1-mm thickness of the anterior (AP) and ventral prostate (VP) of the lightsheet imaging are shown. (F,G) 3D fluorescence imaging of lymphatic vessels in the mouse prostate. The intact prostate of the 8-week-old male mouse was subjected to the whole-tissue VEGFR3-immunolabeling. (F) Representative 3D projection of the lightsheet imaging is shown. (G) Representative optical sections (upper panels) and 3D projections (lower panels) of the 1-mm thickness of the anterior (AP) and ventral prostate (VP) of the lightsheet imaging are shown.

FIGURE 2

3D assessment of neural innervations in the human prostate. (A) The unsectioned prostate tissue of the adult human male before (upper panel) and after (lower panel) the whole-tissue immunolabeling and optical clearing. (B,C) 3D fluorescence imaging of neural innervations in the human prostate. The unsectioned prostate tissue of the adult human male was subjected to the whole-tissue PGP9.5-immunolabeling. (B) Representative optical section (left panel) and 3D projection (right panel) of the lightsheet imaging are shown. The squares denote the capsule or the gland region imaged at high magnification in panel (C). (C) Representative 3D projections of the 1-mm thickness of the capsule (left panel) or the gland region (right panel) of the lightsheet imaging are shown. (D,E) 3D assessment of sympathetic innervations in the human prostate. The unsectioned prostate tissue of the adult human male was subjected to the whole-tissue TH-immunolabeling. (D) Representative optical section (left panel) and 3D projection (right panel) of the lightsheet imaging are shown. The squares denote the capsule or the gland region imaged at high magnification in panel (E). (E) Representative 3D projections of the 1-mm thickness of the capsule (left panel) or the gland region (right panel) of the lightsheet imaging are shown.

FIGURE 3

Autonomic innervations were unaffected in the mouse prostate after castration. Eight-week-old male mice were castrated. Four weeks after castration, the intact prostates were harvested and subjected to the whole-tissue immunolabeling of PGP9.5 (A,B), TH (C,D), or VAChT (E,F). (A,C,E) Representative 3D projections of the lightsheet imaging are shown. (B,D,F) Representative 3D projections of the 1-mm thickness of the anterior (AP) and ventral prostate (VP) of the lightsheet imaging are shown. (G) Tissue volumes of the anterior (AP) and ventral prostate (VP) of the mice that underwent castration or sham surgery were quantified. n = 4, mean ± SEM, *p < 0.05 (two-way ANOVA test). (H) The density of PGP9.5-positive total axons, TH-positive sympathetic axons, or VAChT-positive parasympathetic axons in the anterior (AP) and ventral prostate (VP) was quantified. n = 4, mean ± SEM, *p < 0.05 (two-way ANOVA test). (I) The total length of PGP9.5-positive axons, TH-positive sympathetic axons, or VAChT-positive parasympathetic axons in the anterior (AP) and ventral prostate (VP) was calculated. n = 4, mean ± SEM, n.s., not significant (two-way ANOVA test).

FIGURE 4

Pharmacologic destruction of sympathetic innervations in the mouse prostate caused sterile inflammation. Eight-week-old male mice were intraperitoneally treated with 6-hydroxydopamine or saline, and the prostates were harvested 2 weeks after the treatment. (A,B) The intact prostates were subjected to the whole-tissue TH-immunolabeling. (A) Representative 3D projections of the lightsheet imaging are shown. (B) Representative 3D projections of the 1-mm thickness of the anterior (AP) and ventral prostate (VP) of the lightsheet imaging are shown. (C) Tissue volumes of the anterior (AP) and ventral prostate (VP) of the saline-treated or 6-OHDA-treated mice were quantified. n = 4, mean ± SEM, n.s., not significant (two-way ANOVA test). (D) TH-positive sympathetic axons or VAChT-positive parasympathetic axons in the anterior (AP) and ventral prostate (VP) were quantified. n = 4, mean ± SEM, *p < 0.05, n.s., not significant (two-way ANOVA test). (E) mRNA levels of pro-inflammatory cytokines and chemokines in the anterior (AP) and ventral prostate (VP) were analyzed by the qPCR analysis. n = 4, mean ± SEM, *p < 0.05 (two-way ANOVA test). (F) Cytokines and chemokines in the anterior prostate were analyzed by the mouse cytokine array. (G) The mice at 2 weeks after the 6-OHDA treatment were daily treated by antibiotics via intraperitoneal injection for 5 days. mRNA levels of pro-inflammatory cytokines and chemokines in the anterior prostate (AP) were determined by the qPCR analysis. n = 5, mean ± SEM, n.s., not significant (one-way ANOVA test).

FIGURE 5

Genetic ablation of sympathetic innervations produced sterile inflammation in the prostate. The prostates of Th-Cre; TrkA^fl/fl and control Th-Cre; TrkA^+/+ mice of 8 weeks old were harvested. (A,B) The intact prostates were subjected to the whole-tissue immunolabeling of TH (A) or VAChT (B). Representative 3D projections of the lightsheet imaging are shown. (C) Tissue volumes of the anterior (AP) and ventral prostate (VP) of Th-Cre; TrkA^fl/fl and control Th-Cre; TrkA^+/+ mice were quantified. n = 4, mean ± SEM, n.s., not significant (two-way ANOVA test). (D) TH-positive sympathetic axons or VAChT-positive parasympathetic axons in the anterior (AP) and ventral prostate (VP) were quantified. n = 4, mean ± SEM, *p < 0.05, n.s., not significant (two-way ANOVA test). (E) mRNA levels of pro-inflammatory cytokines and chemokines in the anterior (AP) and ventral prostate (VP) of Th-Cre; TrkA^fl/fl and control Th-Cre; TrkA^+/+ mice were analyzed by the qPCR analysis. n = 4, mean ± SEM, *p < 0.05 (two-way ANOVA test).

FIGURE 6

β2-Adrenergic receptor signal suppressed the prostate inflammation caused by sympathetic loss. (A) Expression profile of adrenergic receptors in the mouse prostate. mRNA levels of adrenergic receptors in the prostates of 8-week-old male mice were analyzed by the qPCR analysis. n = 3, mean ± SEM. (B) The α1-adrenergic receptor antagonist did not cause prostate inflammation. Eight-week-old male mice were daily administered with prazosin via intraperitoneal injection for 14 days. mRNA levels of pro-inflammatory cytokines and chemokines in the anterior prostate (AP) were analyzed by the qPCR analysis. n = 4, mean ± SEM, n.s., not significant (Student’s t-test). (C,D) 8-week-old male mice were intraperitoneally treated with 6-hydroxydopamine or saline control. (C) The β2-adrenergic receptor agonists suppressed prostate inflammation caused by sympathetic loss. The mice at 2 weeks after the 6-OHDA treatment were daily administered with the α1-adrenergic receptor agonist phenylephrine or β2-adrenergic receptor agonists clenbuterol or formoterol via intraperitoneal injection for 5 days. mRNA levels of pro-inflammatory cytokines and chemokines in the anterior prostate (AP) were analyzed by the qPCR analysis. n = 4, mean ± SEM, *p < 0.05, n.s., not significant (one-way ANOVA test). (D) Increased accumulation of CD11b⁺F4/80⁺ macrophages during prostate inflammation. Immune cells in the anterior prostate at 2 weeks after the 6-OHDA treatment were examined by the FACS analysis. n = 5, mean ± SEM, *p < 0.05 (Student’s t-test). (E) Expression profile of adrenergic receptors in CD11b⁺F4/80⁺ macrophages of the prostate. mRNA levels of adrenergic receptors in the CD11b⁺F4/80⁺ macrophages FACS-sorted from the prostate were analyzed by the qPCR analysis. n = 2, mean.