Inflammation Impacts Androgen Receptor Signaling in Basal Prostate Stem Cells Through Interleukin 1 Receptor Antagonist

Abstract

The majority of patients with benign prostate hyperplasia (BPH) exhibit chronic prostate inflammation and the extent of inflammation correlates with the severity of symptoms. How inflammation contributes to prostate enlargement and/or BPH symptoms and the underlying mechanisms are not clearly understood. We established a unique mouse model Prostate Ovalbumin Expressing Transgenic 3 (POET3) that mimics chronic non-bacterial prostatitis in men to study the role of inflammation in prostate hyperplasia. After the injection of ovalbumin peptide-specific T cells, POET3 prostates exhibited an influx of inflammatory cells and an increase in pro-inflammatory cytokines that led to epithelial and stromal hyperplasia. We have previously demonstrated with the POET3 model that inflammation expands the basal prostate stem cell (bPSC) population and promotes bPSC differentiation in organoid cultures. In this study, we investigated the mechanisms underlying the impact of inflammation on bPSC. We found that AR activity was enhanced in inflamed bPSC and was essential for bPSC differentiation in organoid cultures. Most importantly, we identified, for the first time, interleukin 1 receptor antagonist (IL-1RA) as a key regulator of AR in basal stem cells. IL-1RA was one of the top genes upregulated by inflammation and inhibition of IL-1RA abrogated the enhanced AR nuclear accumulation and activity in organoids derived from inflamed bPSC. The mirroring effects of IL-1RA recombinant protein and IL-1α neutralizing antibody suggest that IL-1RA may function by antagonizing IL-1α inhibition of AR expression. Furthermore, we established a lineage tracing model to follow bPSC during inflammation and under castrate conditions. We found that inflammation induced bPSC proliferation and differentiation into luminal cells even under castrate conditions, indicating that AR activation driven by inflammation in bPSC is sufficient for their proliferation and differentiation under androgen-deprived conditions. However, proliferation of the differentiated bPSC in the luminal layer significantly diminished with castration, suggesting inflammation may not maintain AR activity in stromal cells, as stromal cells deprived of androgen after castration could no longer provide paracrine growth factors essential for luminal proliferation. Taken together, we have discovered novel mechanisms through which inflammation modulates AR signaling in bPSC and induces bPSC luminal differentiation that contributes to prostate hyperplasia.

Keywords: androgen receptor; inflammation; interleukin 1 receptor antagonist; prostate; stem cells.

Figure 1.. AR expression and activity are elevated in bPSC freshly isolated from inflamed prostates and the enhanced AR activity was sustained in inflamed organoids in the absence of androgen.

(A) Representative flow cytometry dot plots and histograms of intracellular AR in bPSC from naïve and inflamed prostates. (B) Bar graph showing the percentage of AR+ cells within naïve and inflamed bPSC population as analyzed with flow cytometry (Naïve, n=10; Inflamed, n=5). (C) Immunoblot of full-length AR in naïve and inflamed bPSC lysates. (D) Fold change of AR target genes significantly upregulated in inflamed bPSC (Naïve or Inflamed, n=3). (E) qRT-PCR of conventional AR target genes, Tmprss2 and Nkx3–1, in bPSC. Matched luminal population is included as a control (Naïve or Inflamed, n=3). (F) Immunoblot showing increased level of full-length AR in inflamed organoids compared to naïve organoids. Full-length AR levels in both naïve and inflamed organoids were elevated with 1 nM R1881 treatment. (G) AR Immunofluorescence staining in organoids derived from naïve or inflamed bPSC. Representative cells with nuclear localized AR are indicated with white arrows AR nuclear localization is also shown in the inset image with higher magnification (Scale bar, 100 μm). (H) Fold change of AR target genes significantly upregulated in inflamed organoids (Naïve or Inflamed, n=3).

Figure 2.. Abrogation of AR activity inhibits the differentiation of inflamed bPSC in organoid cultures.

(A) Number of organoids formed with naïve, inflamed bPSC with or without 10 μM Enz treatment (Naïve groups, n=10; Inflamed groups, n=18). (B) Diameter of organoids and (C) percentage of organoids with tubule-like structures in naïve, inflamed organoids or inflamed organoids treated with Enz (Naïve, n=74; Inflamed, n=47; Inflamed+Enz, n=25). (D) Representative H&E images of naïve, inflamed organoids or inflamed organoids treated with Enz (Scale bar, 100 μm). (E) CK5 (green) and CK8 (red) staining showing lack of organization in Enz-treated inflamed organoids (Scale bar, 25 μm). (F) Enz significantly reduces the number of organoids formed with human PSC (control or Enz, n=27).

Figure 3.. IL-1RA is upregulated in inflamed bPSC.

(A) Il1rn and Il1a (arrows) are among the most upregulated genes in the inflamed bPSC compared to naïve bPSC as analyzed with scRNA-seq. (B) Violin plots of Il1rn, Il1a and Il1r1 expression in naïve and inflamed bPSC. (C) qRT-PCR of Il1rn isoforms and Il1a in naïve and inflamed bPSC (Naïve, n=3–4; Inflamed, n=3–8). (D) Upregulation of IL-1RA protein is confirmed using ELISA with lysates collected from naïve or inflamed bPSC (Naïve or Inflamed, n=4).

Figure 4.. IL-1RA mediates inflammation-induced differentiation and AR activity in bPSC organoids.

(A) Representative H&E images of naïve organoids treated with recombinant IL-1RA or neutralizing antibody (Ab) against IL-1α, showing an increase in stratification of treated organoids (Scale bar, 50 μm). (B) Organoid formation with naïve bPSC was significantly increased with recombinant IL-1RA (50 ng/mL) or IL-1α neutralizing Ab treatment (5 μg/mL) (Naïve_control, n=12; Naïve_IL-1RA, n=12; Naïve_IgG, n=6; Naïve_IL-1α Ab, n=9). (C) Quantitation of AR+ (both overall and nuclear) cells in naïve or inflamed organoids treated with IL-1RA (50 ng/mL) (All groups, n=5). (D) AR staining (overall and nuclear) was increased in naïve organoids treated with an IL-1α neutralizing Ab (5 μg/mL) and decreased in inflamed organoids treated with an IL-1RA neutralizing Ab (5 μg/mL) (All groups, n=5). (E) qRT-PCR showing the AR target gene Steap4 was upregulated with IL-1RA in naïve organoids and Enz treatment abolished the upregulation (All groups, n=3). (F) The effects of IL-1RA were lost with naïve organoids derived from Ar_flox/y bPSC which were deprived of AR (All groups, n=9).

Figure 5.. Inflammation mimicking non-bacterial prostatitis promotes the proliferation of basal stem cells and the basal to luminal differentiation in vivo.

(A) Quantitation of BrdU+ bPSC in naïve and inflamed prostates, with or without castration (All groups, n=4). (B) Percentage of bPSC in naïve and inflamed prostates, with or without castration (All groups, n=4). (C) Experimental scheme showing time points of inflammation, castration and analysis. (D) AR staining shows maintenance of AR nuclear localization (arrow heads) in castrated inflamed prostates (Scale bar, 20 μm). (E,F) Quantitation of GFP+ cells or foci within the luminal layer (Naïve, n=3; Naïve_castration, n=4; Inflamed, n=6; Inflamed_castration, n=3). (G) Representative immunofluorescent images from naïve and inflamed prostates with or without castration showing the presence of GFP+CK5+ cells within the CK8+ luminal layer (arrowheads) (Scale bar, 50 μm). (H,I) Size of GFP+ foci and percentage of GFP+ foci of different sizes within the luminal layer (Naïve, n=3; Naïve_castration, n=4; Inflamed, n=6; Inflamed_castration, n=3).

Figure 6.. Diagram of enhanced AR signaling, bPSC proliferation and differentiation that are mediated through IL-1RA during prostate inflammation that mimics the non-bacterial prostatitis.

(This image was made at Biorender.com)