Unraveling CCL20's role by regulating Th17 cell chemotaxis in experimental autoimmune prostatitis

Abstract

Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS), a prevalent urological ailment, exerts a profound influence upon the well-being of the males. Autoimmunity driven by Th17 cells has been postulated as a potential factor in CP/CPPS pathogenesis. Nonetheless, elucidating the precise mechanisms governing Th17 cell recruitment to the prostate, triggering inflammation, remained an urgent inquiry. This study illuminated that CCL20 played a pivotal role in attracting Th17 cells to the prostate, thereby contributing to prostatitis development. Furthermore, it identified prostate stromal cells and immune cells as likely sources of CCL20. Additionally, this research unveiled that IL-17A, released by Th17 cells, could stimulate macrophages to produce CCL20 through the NF-κB/MAPK/PI3K pathway. The interplay between IL-17A and CCL20 establishes a positive feedback loop, which might serve as a critical mechanism underpinning the development of chronic prostatitis, thus adding complexity to its treatment challenges.

Keywords: CCL20/CCR6 axis; IL‐17A; MAPK; NF‐κB; PI3K; Th17; experimental autoimmune prostatitis.

FIGURE 1

Experimental autoimmune prostatitis (EAP) induction led to pelvic discomfort, histological alterations in prostate tissues, heightened activity of CD4 + IL‐17A+ cells in splenic lymphocytes and elevated levels of IL‐17 in the serum. (A–C) Pelvic pain induced by EAP was evaluated through von Frey testing for tactile allodynia in NOD mice. This involved measuring the response frequencies to mechanical filament stimulation of the pelvic region. (D) Representative images of HE staining. (E) Histological assessment to determine the extent of inflammation. (F, G) Flow cytometry staining of splenic lymphocytes to detect CD4 + IL‐17A+ cells. (H) Quantitative analysis of inflammatory cytokines (IL‐17A, IL‐17F, IL‐22, IFN‐γ, TNF‐α and GM‐CSF) in the serum of mice determined by ELISA. Representative data from three independent experiments are shown. Data were presented as mean ± SD and were analysed using one‐way anova analysis (A–C), unpaired, two‐tailed Student's t‐test analysis (G, H) or Wilcoxon rank‐sum test (E). ‘N.D.’ Not Determined; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

The increased infiltration of Th17 cells in the prostate in experimental autoimmune prostatitis mice. (A) Immunohistochemical staining of IL‐17A (marker of Th17) on paraffin‐embedded prostate sections. (B) Semi‐quantitative analysis of IL‐17A in Immunohistochemical staining. (C, D) Flow cytometry analysis of CD4 + IL‐17A+ cells in prostate tissues. (E, F) The IL‐17A expression in prostate tissues assessed through Western Blot analysis. (G) Relative expression of IL‐17A in the prostate measured through RT‐qPCR. (H) Quantitative analysis of inflammatory cytokines (IL‐17A, IL‐17F, IL‐22, IFN‐γ, TNF‐α, GM‐CSF) in the prostate of mice determined by ELISA. Representative data from three independent experiments are shown. Data were presented as mean ± SD and were analysed using unpaired, two‐tailed Student's t‐test analysis. ‘N.D.’ Not Determined; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

The increased levels of CCL20 in the prostate in experimental autoimmune prostatitis mice. (A) Immunohistochemical staining of CCL20 on paraffin‐embedded prostate sections. (B) Semi‐quantitative analysis of CCL20 in Immunohistochemical staining. (C) Relative expression of CCL20 and CCR6 in the prostate measured by RT‐qPCR. (D–F) The expression level of CCL20 and CCR6 in the prostate tissues assessed by Western Blot. (G, H) Quantitative analysis of CCL20 in the serum and prostate of mice determined by ELISA. Representative data from three independent experiments are shown. Data were presented as mean ± SD and were analysed using unpaired, two‐tailed Student's t‐test analysis. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

The recruitment of Th17 cells by CCL20. (A, D) Counting Th17 cells chemoattracted by CCL20 through transwell assay. (B) Immunohistochemical staining of IL‐17A on paraffin‐embedded prostate sections in each group. (C) Assessment of cell viability of Th17 cells after treatment with rCCL20 or anti‐CCL20 neutralizing antibodies. (E) Semi‐quantitative analysis of CCL20 in Immunohistochemical staining. (F) Quantitative analysis of IL‐17A in the serum of mice determined by ELISA. (G, H) Flow cytometry analysis of CD4⁺IL‐17A⁺ cells in the prostate. (I–K) The expression level of IL‐17A and CCR6 in the prostate tissues determined by Western Blot. (L) Relative expression of IL‐17A and CCR6 in the prostate measured by RT‐qPCR. Representative data from three independent experiments are shown. (M, N) Quantitative analysis of inflammatory cytokines (IL‐17A, IL‐17F, IL‐22, IFN‐γ, TNF‐α, GM‐CSF) in the serum and prostate of mice determined by ELISA. Data were presented as mean ± SD and were analysed using one‐way anova analysis. ‘N.D.’ Not Determined; ‘ns’ p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

The potential source CCL20 in the prostate of experimental autoimmune prostatitis (EAP) mice. Representative photographs of immunofluorescence staining for CCL20 and markers for (A) CD4⁺ T cells, (B) macrophages and (C, D) prostatic stromal cells of EAP mice. (E) Quantification of CCL20 immunofluorescence intensity in the experiments of (A–D). Representative data from three independent experiments are shown. Data were presented as mean ± SD and were analysed using one‐way anova analysis. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

IL‐17A promoted secretion of CCL20 of macrophages via the NF‐κB/MAPK/PI3K pathway. (A, B) Western blot analysis of CCL20 and the phosphorylation of the PI3K (mTOR and AKT) NF‐κB (P65) and MAPK (ERK1/2, P38) pathways in BMDM and RAW264.7 after treatment of LPS and/or rIL‐17. (C, D) Quantitative analysis of CCL20 in the supernatant of BMDM and RAW264.7 determined by ELISA. (E, F) Relative expression of CCL20 in BMDM and RAW264.7 measured by RT‐qPCR after treatment of LPS and/or rIL‐17A. (G, H) Western blot analysis of CCL20 after injection of rIL‐17A or anti‐IL‐17A neutralizing antibodies. (I, J) Quantitative analysis of CCL20 in the serum and prostate of mice determined by ELISA. Representative data from three independent experiments are shown. Data were presented as mean ± SD and were analysed using one‐way anova analysis. ‘ns’ p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.